Solid support for mass spectrometry

ABSTRACT

Fast and highly accurate mass spectrometry-based processes for detecting particular nucleic acid molecules and sequences in the molecules are provided. Arrays of oligonucleotides for performing mass spectrometric analyses are provided. In one aspect, a solid support is provided that includes a plurality of single-stranded oligonucleotides immobilized thereon by a linker cleavable under conditions of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry; and matrix for performing MALDI mass spectrometry, wherein the support comprises a flat surface.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/796,416, filed Feb. 28, 2001, now U.S. Pat. No. 6,500,621 which is acontinuation of U.S. application Ser. No. 09/495,444, filed Jan. 31,2000, now U.S. Pat. No. 6,300,076, and of U.S. application Ser. No.09/504,245, filed Feb. 15, 2000, now U.S. Pat. No. 6,221,605. Thisapplication also is a continuation of U.S. application Ser. No.09/287,679, filed Apr. 6, 1999, now U.S. Pat. No. 6,258,538. Thisapplication is also a continuation of U.S. application Ser. No.08/617,256, filed Mar. 18, 1996 and now U.S. Pat. No. 6,043,031. Thisapplication is also a continuation-in-part of U.S. application Ser. No.08/406,199 filed Mar.17, 1995, and now U.S. Pat. No. 5,605,798. U.S.application Ser. No. 08/406,199. The subject matter of each of theseapplications is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The genetic information of all living organisms (e.g. animals, plantsand microorganisms) is encoded in deoxyribonucleic acid (DNA). Inhumans, the complete genome is comprised of about 100,000 genes locatedon 24 chromosomes (The Human Genome, T. Strachan, BIOS ScientificPublishers, 1992). Each gene codes for a specific protein which afterits expression via transcription and translation, fulfills a specificbiochemical function within a living cell. Changes in a DNA sequence areknown as mutations and can result in proteins with altered or in somecases even lost biochemical activities; this in turn can cause geneticdisease. Mutations include nucleotide deletions, insertions oralterations (i.e. point mutations). Point mutations can be either“missense”, resulting in a change in the amino acid sequence of aprotein or “nonsense” coding for a stop codon and thereby leading to atruncated protein.

More than 3000 genetic diseases are currently known (Human GenomeMutations, D. N. Cooper and M. Krawczak, BIOS Publishers, 1993),including hemophilias, thalassemias, Duchenne Muscular Dystrophy (DMD),Huntington's Disease (HD), Alzheimer's Disease and Cystic Fibrosis (CF).In addition to mutated genes, which result in genetic disease, certainbirth defects are the result of chromosomal abnormalities such asTrisomy 21 (Down's Syndrome), Trisomy 13 (Patau Syndrome), Trisomy 18(Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY). Further,there is growing evidence that certain DNA sequences may predispose anindividual to any of a number of diseases such as diabetes,arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g.colorectal, breast, ovarian, lung).

Viruses, bacteria, fungi and other infectious organisms contain distinctnucleic acid sequences, which are different from the sequence containedin the host cell. Therefore, infectious organisms can also be detectedand identified based on their specific DNA sequences.

Since the sequence of about 16 nucleotides is specific on statisticalgrounds even for the size of the human genome, relatively short nucleicacid sequences can be used to detect normal and defective genes inhigher organisms and to detect infectious microorganisms (e.g. bacteria,fungi, protists and yeast) and viruses. DNA sequences can even serve asa fingerprint for detection of different individuals within the samespecies. (Thompson, J. S. and M. W. Thompson, eds., Genetics inMedicine, W. B. Saunders Co., Philadelphia, Pa. (1986).

Several methods for detecting DNA are currently being used. For example,nucleic acid sequences can be identified by comparing the mobility of anamplified nucleic acid fragment with a known standard by gelelectrophoresis, or by hybridization with a probe, which iscomplementary to the sequence to be identified. Identification, however,can only be accomplished if the nucleic acid fragment is labeled with asensitive reporter function (e.g. radioactive (³²P, ³⁵S), fluorescent orchemiluminescent). However, radioactive labels can be hazardous and thesignals they produce decay over time. Non-isotopic labels (e.g.fluorescent) suffer from a lack of sensitivity and fading of the signalwhen high intensity lasers are being used. Additionally, performinglabeling, electrophoresis and subsequent detection are laborious,time-consuming and error-prone procedures. Electrophoresis isparticularly error-prone, since the size or the molecular weight of thenucleic acid cannot be directly correlated to the mobility in the gelmatrix. It is known that sequence specific effects, secondary structuresand interactions with the gel matrix are causing artifacts.

In general, mass spectrometry provides a means of “weighing” individualmolecules by ionizing the molecules in vacuo and making them “fly” byvolatilization. Under the influence of combinations of electric andmagnetic fields, the ions follow trajectories depending on theirindividual mass (m) and charge (z). In the range of molecules with lowmolecular weight, mass spectrometry has long been part of the routinephysical-organic repertoire for analysis and characterization of organicmolecules by the determination of the mass of the parent molecular ion.In addition, by arranging collisions of this parent molecular ion withother particles (e.g. argon atoms), the molecular ion is fragmentedforming secondary ions by the so-called collision induced dissociation(CID). The fragmentation pattern/pathway very often allows thederivation of detailed structural information. Many applications of massspectrometric methods are known in the art, particularly in biosciences,and can be found summarized in Methods of Enzymology, Vol. 193:“MassSpectrometry” (J. A. McCloskey, editor), 1990, Academic Press, New York.

Due to the apparent analytical advantages of mass spectrometry inproviding high detection sensitivity, accuracy of mass measurements,detailed structural information by CID in conjunction with an MS/MSconfiguration and speed, as well as on-line data transfer to a computer,there has been considerable interest in the use of mass spectrometry forthe structural analysis of nucleic acids. Recent reviews summarizingthis field include K. H. Schram, “Mass Spectrometry of Nucleic AcidComponents, Biomedical Applications of Mass Spectrometry” 34, 203-287(1990); and P. F. Crain, “Mass Spectrometric Techniques in Nucleic AcidResearch, “Mass Spectrometry Reviews 9, 505-554 (1990).

However, nucleic acids are very polar biopolymers that are verydifficult to volatilize. Consequently, mass spectrometric detection hasbeen limited to low molecular weight synthetic oligonucleotides bydetermining the mass of the parent molecular ion and through this,confirming the already known oligonucleotide sequence, or alternatively,confirming the known sequence through the generation of secondary ions(fragment ions) via CID in the MS/MS configuration utilizing, inparticular, for the ionization and volatilization, the method of fastatomic bombardment (FAB mass spectrometry) or plasma desorption (PD massspectrometry). As an example, the application of FAB to the analysis ofprotected dimeric blocks for chemical synthesis of oligodeoxynucleotideshas been described (Wolter et al. Biomedical Environmental MassSpectrometry 14: 111-116 (1987)).

Two more recent ionizations/desorption techniques areelectrospray/ionspray (ES) and matrix-assisted laserdesorption/ionization (MALDI). ES mass spectrometry has been introducedby Yamashita et al. (J. Phys. Chem. 88: 4451-59 (1984); PCT ApplicationNo. WO 90/14148) and current applications are summarized in recentreview articles (R. D. Smith et al., Anal. Chem. 62: 882-89 (1990) andB. Ardrey, Electrospray Mass Spectrometry, Spectroscopy Europe 4: 10-18(1992)). The molecular weights of a tetradecanucleotide (Covey et al.“The Determination of Protein, Oligonucleotide and Peptide MolecularWeights by Ionspray Mass Spectrometry,” Rapid Communications in MassSpectrometry 2: 249-256 (1988)), and of a 21-mer (Methods in Enzymology193, “Mass Spectrometry” (McCloskey, editor), p. 425, 1990, AcademicPress, New York) have been published. As a mass analyzer, a quadrupoleis most frequently used. The determination of molecular weights infemtomole amounts of sample is very accurate due to the presence ofmultiple ion peaks which all could be used for the mass calculation.

MALDI mass spectrometry, in contrast, can be particularly attractivewhen a time-of-flight (TOF) configuration is used as a mass analyzer.The MALDI-TOF mass spectrometry has been introduced by Hillenkamp et al.(“Matrix Assisted UV-Laser Desorption/Ionization: A New Approach to MassSpectrometry of Large Biomolecules,” Biological Mass Spectrometry(Burlingame and McCloskey, editors), Elsevier Science Publishers,Amsterdam, pp. 49-60, 1990). Since, in most cases, no multiple molecularion peaks are produced with this technique, the mass spectra, inprinciple, look simpler compared to ES mass spectrometry.

Although DNA molecules up to a molecular weight of 410,000 daltons havebeen desorbed and volatilized (Nelson et al., “Volatilization of HighMolecular Weight DNA by Pulsed Laser Ablation of Frozen AqueousSolutions, Science 246: 1585-87 (1989)), this technique has so far onlyshown very low resolution (oligothymidylic acids up to 18 nucleotides,Huth-Fehre et al., Rapid Communications in Mass Spectrometry 6: 209-13(1992); DNA fragments up to 500 nucleotidase in length, Tang, K. et al.,Rapid Communications in Mass Spectrometry 8: 727-730 (1994); and adouble-stranded DNA of 28 base pairs (Williams et al., “Time-of FlightMass Spectrometry of Nucleic Acids by Laser Ablation and Ionization froma Frozen Aqueous Matrix,” Rapid Communications in Mass Spectrometry 4:348-351 (1990)).

Japanese Patent No. 59-131909 describes an instrument, which detectsnucleic acid fragments separated either by electrophoresis, liquidchromatography or high speed gel filtration. Mass spectrometricdetection is achieved by incorporating into the nucleic acids, atomswhich normally do not occur in DNA such as S, Br, I or Ag, Au, Pt, Os,Hg.

SUMMARY OF THE INVENTION

The instant invention provides mass spectrometric processes fordetecting a particular nucleic acid sequence in a biological sample.Depending on the sequence to be detected, the processes can be used, forexample, to diagnose (e.g. prenatally or postnatally) a genetic diseaseor chromosomal abnormality; a predisposition to a disease or condition(e.g. obesity, atherosclerosis, cancer), or infection by a pathogenicorganism (e.g. virus, bacteria, parasite or fungus); or to provideinformation relating to identity, heredity, or compatibility (e.g. HLAphenotyping).

In a first embodiment, a nucleic acid molecule containing the nucleicacid sequence to be detected (i.e. the target) is initially immobilizedto a solid support. Immobilization can be accomplished, for example,based on hybridization between a portion of the target nucleic acidmolecule, which is distinct from the target detection site and a capturenucleic acid molecule, which has been previously immobilized to a solidsupport. Alternatively, immobilization can be accomplished by directbonding of the target nucleic acid molecule and the solid support.Preferably, there is a spacer (e.g. a nucleic acid molecule) between thetarget nucleic acid molecule and the support. A detector nucleic acidmolecule (e.g. an oligonucleotide or oligonucleotide mimetic), which iscomplementary to the target detection site can then be contacted withthe target detection site and formation of a duplex, indicating thepresence of the target detection site can be detected by massspectrometry. In preferred embodiments, the target detection site isamplified prior to detection and the nucleic acid molecules areconditioned. In a further preferred embodiment, the target detectionsequences are arranged in a format that allows multiple simultaneousdetections (multiplexing), as well as parallel processing usingoligonucleotide arrays (“DNA chips”).

In a second embodiment, immobilization of the target nucleic acidmolecule is an optional rather than a required step. Instead, once anucleic acid molecule has been obtained from a biological sample, thetarget detection sequence is amplified and directly detected by massspectrometry. In preferred embodiments, the target detection site and/orthe detector oligonucleotides are conditioned prior to massspectrometric detection. In another preferred embodiment, the amplifiedtarget detection sites are arranged in a format that allows multiplesimultaneous detections (multiplexing), as well as parallel processingusing oligonucleotide arrays (“DNA chips”).

In a third embodiment, nucleic acid molecules which have been replicatedfrom a nucleic acid molecule obtained from a biological sample can bespecifically digested using one or more nucleases (usingdeoxyribonucleases for DNA or ribonucleases for RNA) and the fragmentscaptured on a solid support carrying the corresponding complementarysequences. Hybridization events and the actual molecular weights of thecaptured target sequences provide information on whether and wheremutations in the gene are present. The array can be analyzed spot byspot using mass spectrometry. DNA can be similarly digested using acocktail of nucleases including restriction endonucleases. In apreferred embodiment, the nucleic acid fragments are conditioned priorto mass spectrometric detection.

In a fourth embodiment, at least one primer with 3′ terminal basecomplementarity to a an allele (mutant or normal) is hybridized with atarget nucleic acid molecule, which contains the allele. An appropriatepolymerase and a complete set of nucleoside triphosphates or only one ofthe nucleoside triphosphates are used in separate reactions to furnish adistinct extension of the primer. Only if the primer is appropriatelyannealed (i.e. no 3′ mismatch) and if the correct (i.e. complementary)nucleotide is added, will the primer be extended. Products can beresolved by molecular weight shifts as determined by mass spectrometry.

In a fifth embodiment, a nucleic acid molecule containing the nucleicacid sequence to be detected (i.e. the target) is initially immobilizedto a solid support. Immobilization can be accomplished, for example,based on hybridization between a portion of the target nucleic acidmolecule, which is distinct from the target detection site and a capturenucleic acid molecule, which has been previously immobilized to a solidsupport. Alternatively, immobilization can be accomplished by directbonding of the target nucleic acid molecule and the solid support.Preferably, there is a spacer (e.g. a nucleic acid molecule) between thetarget nucleic acid molecule and the support. A nucleic acid moleculethat is complementary to a portion of the target detection site that isimmediately 5′ of the site of a mutation is then hybridized with thetarget nucleic acid molecule. The addition of a complete set ofdideoxynucleosides or 3′-deoxynucleoside triphosphates (e.g. pppAdd,pppTdd, pppCdd and pppGdd) and a DNA dependent DNA polymerase allows forthe addition only of the one dideoxynucleoside or 3′-deoxynucleosidetriphosphate that is complementary to X. The hybridization product canthen be detected by mass spectrometry.

In a sixth embodiment, a target nucleic acid is hybridized with acomplementary oligonucleotides that hybridize to the target within aregion that includes a mutation M. The heteroduplex is than contactedwith an agent that can specifically cleave at an unhybridized portion(e.g. a single strand specific endonuclease), so that a mismatch,indicating the presence of a mutation, results in a the cleavage of thetarget nucleic acid. The two cleavage products can then be detected bymass spectrometry.

In a seventh embodiment, which is based on the ligase chain reaction(LCR), a target nucleic acid is hybridized with a set of ligation eductsand a thermostable DNA ligase, so that the ligase educts becomecovalently linked to each other, forming a ligation product. Theligation product can then be detected by mass spectrometry and comparedto a known value. If the reaction is performed in a cyclic manner, theligation product obtained can be amplified to better facilitatedetection of small volumes of the target nucleic acid. Selection betweenwildtype and mutated primers at the ligation point can result in adetection of a point mutation.

The processes of the invention provide for increased accuracy andreliability of nucleic acid detection by mass spectrometry. In addition,the processes allow for rigorous controls to prevent false negative orpositive results. The processes of the invention avoid electrophoreticsteps, labeling and subsequent detection of a label. In fact it isestimated that the entire procedure, including nucleic acid isolation,amplification, and mass spec analysis requires only about 2-3 hourstime. Therefore the instant disclosed processes of the invention arefaster and less expensive to perform than existing DNA detectionsystems. In addition, because the instant disclosed processes allow thenucleic acid fragments to be identified and detected at the same time bytheir specific molecular weights (an unambiguous physical standard), thedisclosed processes are also much more accurate and reliable thancurrently available procedures.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1A is a diagram showing a process for performing mass spectrometricanalysis on one target detection site (TDS) contained within a targetnucleic acid molecule (T), which has been obtained from a biologicalsample. A specific capture sequence (C) is attached to a solid support(SS) via a spacer (S). The capture sequence is chosen to specificallyhybridize with a complementary sequence on the target nucleic acidmolecule (T), known as the target capture site (TCS). The spacer (S)facilitates unhindered hybridization. A detector nucleic acid sequence(D), which is complementary to the TDS is then contacted with the TDS.Hybridization between D and the TDS can be detected by massspectrometry.

FIG. 1B is a diagram showing a process for performing mass spectrometricanalysis on at least one target detection site (here TDS 1 and TDS 2)via direct linkage to a solid support. The target sequence (T)containing the target detection site (TDS 1 and TDS 2) is immobilized toa solid support via the formation of a reversible or irreversible bondformed between an appropriate functionality (L′) on the target nucleicacid molecule (T) and an appropriate functionality (L) on the solidsupport. Detector nucleic acid sequences (here D1 and D2), which arecomplementary to a target detection site (TDS 1 or TDS 2) are thencontacted with the TDS. Hybridization between TDS 1 and D1 and/or TDS 2and D2 can be detected and distinguished based on molecular weightdifferences.

FIG. 1C is a diagram showing a process for detecting a wildtype (D^(wt))and/or a mutant (D^(mut)) sequence in a target (T) nucleic acidmolecule. As in FIG. 1A, a specific capture sequence (C) is attached toa solid support (SS) via a spacer (S). In addition the capture sequenceis chosen to specifically interact with a complementary sequence on thetarget sequence (T), the target capture site (TCS) to be detectedthrough hybridization. However, the target detection site (TDS) includesmutation, X, which changes the molecular weight; mutated targetdetection sites can be distinguished from wildtype by mass spectrometry.Preferably, the detector nucleic acid molecule (D) is designed so thatthe mutation is in the middle of the molecule and therefore would notlead to a stable hybrid if the wildtype detector oligonucleotide(D^(wt)) is contacted with the target detector sequence, e.g. as acontrol. The mutation can also be detected if the mutated detectoroligonucleotide (D^(mut)) with the matching base as the mutated positionis used for hybridization. If a nucleic acid molecule obtained from abiological sample is heterozygous for the particular sequence (i.e.contain both D^(wt) and D^(mut)) both D^(wt) and D^(mut) will be boundto the appropriate strand and the mass difference allows both D^(wt) andD^(mut) to be detected simultaneously.

FIG. 2 is a diagram showing a process in which several mutations aresimultaneously detected on one target sequence by employingcorresponding detector oligonucleotides. The molecular weightdifferences between the detector oligonucleotides D1, D2 and D3 must belarge enough so that simultaneous detection (multiplexing) is possible.This can be achieved either by the sequence itself (composition orlength) or by the introduction of mass-modifying functionalities M1-M3into the detector oligonucleotide.

FIG. 3 is a diagram showing still another multiplex detection format. Inthis embodiment, differentiation is accomplished by employing differentspecific capture sequences which are position-specifically immobilizedon a flat surface (e.g., a ‘chip array’). If different target sequencesT1-Tn are present, their capture sites TCS1-TCSn will interact withcomplementary immobilized capture sequences C1-Cn. Detection is achievedby employing appropriately mass differentiated detector oligonucleotidesD1-Dn, which are mass differentiated either by their sequences or bymass modifying functionalities M1-Mn.

FIG. 4 is a diagram showing a format wherein a predesigned targetcapture site (TCS) is incorporated into the target sequence using PCRamplification. Only one strand is captured, the other is removed (e.g.,based on the interaction between biotin and streptavidin coated magneticbeads). If the biotin is attached to primer 1 the other strand can beappropriately marked by a TCS. Detection is as described above throughthe interaction of a specific detector oligonucleotide D with thecorresponding target detection site TDS via mass spectrometry.

FIG. 5 is a diagram showing how amplification (here ligase chainreaction (LCR)) products can be prepared and detected by massspectrometry. Mass differentiation can be achieved by the mass modifyingfunctionalities (M1 and M2) attached to primers (P1 and P4respectively). Detection by mass spectrometry can be accomplisheddirectly (i.e.) without employing immobilization and target capturingsites (TCS)). Multiple LCR reaction can be performed in parallel byproviding an ordered array of capturing sequences (C). This formatallows separation of the ligation products and spot by spotidentification via mass spectrometry or multiplexing if massdifferentiation is sufficient.

FIG. 6A is a diagram showing mass spectrometric analysis of a nucleicacid molecule, which has been amplified by a transcription amplificationprocedure. An RNA sequence is captured via its TCS sequence, so thatwildtype and mutated target detection sites can be detected as above byemploying appropriate detector oligonucleotides (D).

FIG. 6B is a diagram showing multiplexing to detect two different(mutated) sites on the same RNA in a simultaneous fashion usingmass-modified detector oligonucleotides M1-D1 and M2-D2.

FIG. 6C is a diagram of a different multiplexing procedure for detectionof specific mutations by employing mass modified dideoxynucleoside or3′-deoxynucleoside triphosphates and an RNA dependent DNA polymerase.Alternatively, DNA dependent RNA polymerase and ribonucleotidetriphosphates can be employed. This format allows for simultaneousdetection of all four base possibilities at the site of a mutation (X).

FIG. 7A is a diagram showing a process for performing mass spectrometricanalysis on one target detection site (TDS) contained within a targetnucleic acid molecule (T), which has been obtained from a biologicalsample. A specific capture sequence (C) is attached to a solid support(SS) via a spacer (S). The capture sequence is chosen to specificallyhybridize with a complementary sequence on T known as the target capturesite (TCS). A nucleic acid molecule that is complementary to a portionof the TDS is hybridized to the TDS 5′ of the site of a mutation (X)within the TDS. The addition of a complete set of dideoxynucleosides or3′-deoxynucleoside triphosphates (e.g. pppAdd, pppTdd, pppCdd andpppGdd) and a DNA dependent DNA polymerase allows for the addition onlyof the one dideoxynucleoside or 3′-deoxynucleoside triphosphate that iscomplementary to X.

FIG. 7B is a diagram showing a process for performing mass spectrometricanalysis to determine the presence of a mutation at a potential mutationsite (M) within a nucleic acid molecule. This format allows forsimultaneous analysis of both alleles (A) and (B) of a double strandedtarget nucleic acid molecule, so that a diagnosis of homozygous normal,homozygous mutant or heterozygous can be provided. Allele A and B areeach hybridized with complementary oligonucleotides ((C) and (D)respectively), that hybridize to A and B within a region that includesM. Each heteroduplex is then contacted with a single strand specificendonuclease, so that a mismatch at M, indicating the presence of amutation, results in the cleavage of (C) and/or (D), which can then bedetected by mass spectrometry.

FIG. 8 is a diagram showing how both strands of a target DNA can beprepared for detection using transcription vectors having two differentpromoters at opposite locations (e.g. the SP 6 and T7 promoter). Thisformat is particularly useful for detecting heterozygous targetdetection sites (TDS). Employing the SP 6 or the T7 RNA polymerase bothstrands could be transcribed separately or simultaneously. Both RNAs canbe specifically captured and simultaneously detected using appropriatelymass-differentiated detector oligonucleotides. This can be accomplishedeither directly in solution or by parallel processing of many targetsequences on an ordered array of specifically immobilized capturingsequences.

FIG. 9 is a diagram showing how RNA prepared as described in FIGS. 6, 7and 8 can be specifically digested using one or more ribonucleases andthe fragments captured on a solid support carrying the correspondingcomplementary sequences. Hybridization events and the actual molecularweights of the captured target sequences provide information on whetherand where mutations in the gene are present. The array can be analyzedspot by spot using mass spectrometry. DNA can be similarly digestedusing a cocktail of nucleases including restriction endonucleases.Mutations can be detected by different molecular weights of specific,individual fragments compared to the molecular weights of the wildtypefragments.

FIG. 10A shows a spectra resulting from the experiment described in thefollowing Example 1. FIG. 10A-1 shows the absorbance or the 26-merbefore hybridization. FIG. 10A-2 shows the filtrate of thecentrifugation after hybridization. FIG. 10A-3 shows the results afterthe first wash with 50 mM ammonium citrate. FIG. 10A-4 shows the resultsafter the second wash with 50 mM ammonium citrate.

FIG. 10B shows a spectra resulting from the experiment described in thefollowing Example 1 after three washing/centrifugation steps.

FIG. 10C shows a spectra resulting from the experiment described in thefollowing Example 1 showing the successful desorption of the hybridized26-mer off of beads.

FIG. 11 shows a spectra resulting from the experiment described in thefollowing Example 1 showing the successful desorption of the hybridized40-mer. The efficiency of detection suggests that fragments much longer40-mers can also be desorbed.

FIGS. 12A-12C show spectra resulting from the experiment described inthe following Example 2 showing the successful desorption anddifferentiation of an 18-mer and 19-mer by electrospray massspectrometry: the mixture (12A), peaks resulting from the 18-mer,emphasized (12B) and peaks resulting from the 19-mer, emphasized (12C).

FIG. 13 is a graphic representation of the process for detecting theCystic Fibrosis mutation ΔF508 as described in Example 3; N indicatesnormal and M indicats the mutation detection primer or extended primer.

FIG. 14 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal.

FIG. 15 is a mass spectrum of the DNA extension product of a ΔF508heterozygous mutant.

FIG. 16 is a mass spectrum of the DNA extension product of a ΔF508homozygous normal.

FIG. 17 is a mass spectrum of the DNA extension product of a ΔF508homozygous mutant.

FIG. 18 is mass spectrum of the DNA extension product of a ΔF508heterozygous mutant.

FIG. 19 is a graphic representation of various processes for performingapolipoprotein E genotyping.

FIGS. 20A-20B show the nucleic acid sequence of normal apolipoprotein E(encoded by the E3 allele) and other isotypes encoded by the E2 and E4alleles.

FIG. 21A shows the composite restriction pattern for various genotypesof apolipoprotein E.

FIG. 21B shows the restriction pattern obtained in a 3.5% MetPhorAgarose Gel for various genotypes of apolipoprotein E.

FIG. 21C shows the restriction pattern obtained in a 12% polyacrylamidegel for various genotypes of apolipoprotein E.

FIG. 22A is a chart showing the molecular weights of the 91, 83, 72, 48and 35 base pair fragments obtained by restriction enzyme cleavage ofthe E2, E3, and E4 alleles of apolipoprotein E.

FIG. 22B is the mass spectra of the restriction product of a homozygousE4 apolipoprotein E genotype.

FIG. 23A is the mass spectra of the restriction product of a homozygousE3 apolipoprotein E genotype.

FIG. 23B is the mass spectra of the restriction product of a E3/E4apolipoprotein E genotype.

FIG. 24 is an autoradiograph of a 7.5% polyacrylamide gel in which 10%(5 μl) of each PCR was loaded. Sample M: pBR322 Alul digested; sample 1:HBV positive in serological analysis; sample 2: also HBV positive;sample 3: without serological analysis but with an increased level oftransaminases, indicating liver disease; sample 4: HBV negative; sample5: HBV positive by serological analysis; sample 6: HBV negative (−)negative control; (+) positive control). Staining was done with ethidiumbromide.

FIG. 25A is a mass spectrum of sample 1, which is HBV positive. Thesignal at 20754 Da represent HBV related PCR product (67 nucleotides,calculated mass: 20735 Da). The mass signal at 10390 Da represents[M+2H]²⁺ signal (calculated: 10378 Da).

FIG. 25B is a mass spectrum of sample 3, which is HBV negativecorresponding to PCR, serological and dot blot based assays. The PCRproduct is generated only in trace amounts. Nevertheless, it isunambiguously detected at 20751 Da (calculated: 20735 Da). The masssignal at 10397 Da represents the [M+2H]²⁺ molecule ion (calculated:10376 Da).

FIG. 25C is a mass spectrum of sample 4, which is HBV negative, but CMVpositive. As expected, no HBV specific signals could be obtained.

FIG. 26 shows a part of the E.coli lacl gene with binding sites of thecomplementary oligonucleotides used in the ligase chain reaction (LCR).Here the wildtype sequence is displayed. The mutant contains a pointmutation at bp 191 which is also the site of ligation (bold). Themutation is a C to T transition (G to A, respectively). This leads to aT-G mismatch with oligo A (and A-C mismatch with oligo B, respectively).

FIG. 27 is a 7.5% polyacrylamide gel stained with ethidium bromide. M:chain length standard (pUC19 DNA, Mspl digested). Lane 1: LCR withwildtype template. Lane 2: LCR with mutant template. Lane 3: (control)LCR without template. The ligation product (50 bp) was only generated inthe positive reactive containing wildtype template.

FIG. 28 is an HPLC chromatogram of two pooled positive LCRs.

FIG. 29 shows an HPLC chromatogram under the same conditions but usingthe mutant template. The small signal of the ligation product is due toeither template-free ligation of the educts or to a ligation at a (G-T,A-C) mismatch. The ‘false positive’ signal is significantly lower thanthe signal of ligation product with wildtype template depicted in FIG.28. The analysis of ligation educts leads to ‘double-peaks’ because twoof the oligonucleotides are 5′-phosphorylated.

FIG. 30A shows the complex signal pattern obtained by MALDI-TOF-MSanalysis of Pfu DNA-ligase solution. FIG. 30B shows a MALDI-TOF-spectrumof an unpurified LCR. The mass signal 67569 Da probably represents thePfu DNA ligase.

FIG. 31A shows a MALDI-TOF spectrum of two pooled positive LCRs. Thesignal at 7523 Da represents unligated oligo A (calculated: 7521 Da)whereas the signal at 15449 Da represents the ligation product(calculated: 15450 Da). The signal at 3774 Da is the [M+2H]²⁺ signal ofoligo A. The signals in the mass range lower than 2000 Da are due to thematrix ions. The spectrum corresponds to lane 1 in FIG. 27 and to thechromatogram in FIG. 28. FIG. 31B shows a spectrum of two poolednegative LCRs (mutant template). The signal at 7517 Da represents oligoA (calculated: 7521 Da).

FIG. 32 shows a spectrum obtained from two pooled LCRs in which onlysalmon sperm DNA was used as a negative control: only oligo A could bedetected, as expected.

FIG. 33A shows a spectrum of two pooled positive LCRs. The purificationwas done with a combination of ultrafiltration and streptavidinDynaBeads as described in the text. The signal at 15448 Da representsthe ligation product (calculated: 15450 Da). The signal at 7527represents oligo A (calculated: 7521 Da). The signals at 3761 Da is the[M+2H]²⁺ signal of oligo A, where as the signal at 5140 Da is the[M+3H]²⁺ signal of the ligation product. FIG. 33B shows a spectrum oftwo pooled negative LCRs (without template). The signal at 7514 Darepresents oligo A (calculated: 7521 Da).

FIG. 34A is a schematic representation of the oligo base extension ofthe mutation detection primer b using ddTTP. FIG. 34B is a schematicrepresentation of the oligo base extension of the mutation detectionprimer b using ddCTP. The theoretical mass calculation is given inparenthesis. The sequence shown is part of the exon 10 of the CFTR genethat bears the most common cystic fibrosis mutation ΔF508 and more raremutations Δ1507 as well at Ile506Ser.

FIG. 35A is a MALDI-TOF-MS spectra recorded directly from precipitatedoligo base extended primers for mutation detection using ddTTP. FIG. 35Bis a MALDI-TOF-MS spectra recorded directly from precipitated oligo baseextended primers for mutation detection using ddCTP. The spectra on thetop of each panel (ddTTP or ddCTP, respectively) shows the annealedprimer (CF508) without further extension reaction. The template ofdiagnosis is pointed out below each spectra and the observed/expectedmolecular mass are written in parenthesis.

FIG. 36 shows the portion of the sequence of pRFc1 DNA, which was usedas template for PCR amplification of unmodified and 7-deazapurinecontaining 99-mer and 200-mer nucleic acids as well as the sequences ofthe 19-mer primers and the two 18-mer reverse primers.

FIG. 37 shows the portion of the nucleotide sequence of M13mp18 RFI DNA,which was used for PCR amplification of unmodified and 7-deazapurinecontaining 103-mer nucleic acids. Also shown are nucleotide sequences ofthe 17-mer primers used in the PCR.

FIG. 38 shows the result of a polyacrylamide gel electrophoresis of PCRproducts purified and concentrated for MALDI-TOF MS analysis. M: chainlength marker, lane 1: 7-deazapurine containing 99-mer PCR product, lane2: unmodified 99-mer, lane 3: 7-deazapurine containing 103-mer and lane4: unmodified 103-mer PCR product.

FIG. 39: an autoradiogram of polyacrylamide gel electrophoresis of PCRreactions carried out with 5′-[³²P]-labeled primers 1 and 4. Lanes 1 and2: unmodified and 7-deazapurine modified 200-mer (71123 and 39582counts), lanes 3 and 4: unmodified and 7-deazapurine modified 200-mer(71123 and 39582 counts) and lanes 5 and 6: unmodified and 7-deazapurinemodified 99-mer (173216 and 94400 counts).

FIG. 40A shows a MALDI-TOF mass spectrum of the unmodified 103-mer PCRproducts (sum of twelve single shot spectra). The mean value of themasses calculated for the two single strands (31768 u and 31759 u) is31763 u. Mass resolution: 18. FIG. 40B shows a MALDI-TOF mass spectrumof 7-deazapurine containing 103-mer PCR product (sum of three singleshot spectra). The mean value of the masses calculated for the twosingle strands (31727 u and 31719 u) is 31723 u. Mass resolution: 67.

FIG. 41A shows a MALDI-TOF mass spectrum of the unmodified 99-mer PCRproduct (sum of twenty single shot spectra). Values of the massescalculated for the two single strands: 30261 u and 30794 u. FIG. 41Bshows a MALDI-TOF mass spectrum of the 7-deazapurine containing 99-merPCR product (sum of twelve single shot spectra). Values of the massescalculated for the two single strands:

30224 u and 30750 u.

FIG. 42A shows a MALDI-TOF mass spectrum of the unmodified 200-mer PCRproduct (sum of 30 single shot spectra). The mean value of the massescalculated for the two single strands (61873 u and 61595 u) is 61734 u.Mass resolution: 28. FIG. 42B shows a MALDI-TOF mass spectrum of7-deazapurine containing 200-mer PCR product (sum of 30 single shotspectra). The mean value of the masses calculated for the two singlestrands (61772 u and 61514 u) is 61643 u. Mass resolution: 39.

FIG. 43A shows a MALDI-TOF mass spectrum of 7-deazapurine containing100-mer PCR product with ribomodified primers. The mean value of themasses calculated for the two single strands (30529 u and 31095 u) is30812 u. FIG. 43B shows a MALDI-TOF mass spectrum of the PCR-productafter hydrolytic primer-cleavage. The mean value of the massescalculated for the two single strands (25104 u and 25229 u) is 25167 u.The mean value of the cleaved primers (5437 u and 5918 u) is 5677 u.

FIGS. 44A-44D show the MALDI-TOF mass spectrum of the four sequencingladders obtained from a 39-mer template (SEQ. ID. No. 13), which wasimmobilized to streptavidin beads via a 3′ biotinylation. A 14-merprimer (SEQ. ID. No. 14) was used in the sequencing.

FIG. 45 shows a MALDI-TOF mass spectrum of a solid state sequencing of a78-mer template (SEQ. ID. No. 15), which was immobilized to streptavidinbeads via a 3′ biotinylation. A 18-mer primer (SEQ. ID. No. 16) andddGTP were used in the sequencing.

FIG. 46 shows a scheme in which duplex DNA probes with single-strandedoverhang capture specific DNA templates and also serve as primers forsolid state sequencing.

FIG. 47A-47D shows MALDI-TOF mass spectra obtained from a 5′ fluorescentlabeled 23-mer (SEQ. ID. No. 19) annealed to an 3′ biotinylated 18-mer(SEQ. ID. No. 20), leaving a 5-base overhang, which captured a 15-mertemplate (SEQ. ID. No. 21).

FIGS. 48A-48D show a stacking fluorogram of the same products obtainedfrom the reaction described in FIG. 35, but run on a conventional DNAsequencer.

DETAILED DESCRIPTION OF THE INVENTION

In general, the instant invention provides mass spectrometric processesfor detecting a particular nucleic acid sequence in a biological sample.As used herein, the term “biological sample” refers to any materialobtained from any living source (e.g. human, animal, plant, bacteria,fungi, protist, virus). For use in the invention, the biological sampleshould contain a nucleic acid molecule. Examples of appropriatebiological samples for use in the instant invention include: solidmaterials (e.g. tissue, cell pellets, biopsies) and biological fluids(e.g. urine, blood, saliva, amniotic fluid, mouth wash).

Nucleic acid molecules can be isolated from a particular biologicalsample using any of a number of procedures, which are well-known in theart, the particular isolation procedure chosen being appropriate for theparticular biological sample. For example, freeze-thaw and alkalinelysis procedures can be useful for obtaining nucleic acid molecules fromsolid materials; heat and alkaline lysis procedures can be useful forobtaining nucleic acid molecules from urine; and proteinase K extractioncan be used to obtain nucleic acid from blood (Rolff, A et al. PCR:Clinical Diagnostics and Research, Springer (1994).

To obtain an appropriate quantity of nucleic acid molecules on which toperform mass spectrometry, amplification may be necessary. Examples ofappropriate amplification procedures for use in the invention include:cloning (Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989), polymerase chain reaction (PCR)(C. R. Newton and A. Graham, PCR, BIOS Publishers, 1994); ligase chainreaction (LCR) (Wiedmann, M., et al., PCR Methods Appl. Vol. 3, pp.57-64 (1994); F. Barnay, Proc. Natl. Acad. Sci USA 88: 189-93 (1991));strand displacement amplification (SDA) (G. Terrance Walker et al.,Nucleic Acids Res. 22: 2670-77 (1994)); and variations such as RT-PCR(Higuchi, et al., Bio/Technology 11: 1026-1030 (1993)), allele-specificamplification (ASA) and transcription based processes.

To facilitate mass spectrometric analysis, a nucleic acid moleculecontaining a nucleic acid sequence to be detected can be immobilized toa solid support. Examples of appropriate solid supports include beads(e.g. silica gel, controlled pore glass, magnetic, Sephadex/Sepharose,cellulose), flat surfaces or chips (e.g. glass fiber filters, glasssurfaces, metal surface (steel, gold, silver, aluminum, copper andsilicon), capillaries, plastic (e.g. polyethylene, polypropylene,polyamide, polyvinylidenedifluoride membranes or microtiter plates)); orpins or combs made from similar materials comprising beads or flatsurfaces or beads placed into pits in flat surfaces such as wafers (e.g.silicon wafers).

Immobilization can be accomplished, for example, based on hybridizationbetween a capture nucleic acid sequence, which has already beenimmobilized to the support and a complementary nucleic acid sequence,which is also contained within the nucleic acid molecule containing thenucleic acid sequence to be detected (FIG. 1A). So that hybridizationbetween the complementary nucleic acid molecules is not hindered by thesupport, the capture nucleic acid can include a spacer region of atleast about five nucleotides in length between the solid support and thecapture nucleic acid sequence. The duplex formed will be cleaved underthe influence of the laser pulse and desorption can be initiated. Thesolid support-bound base sequence can be presented through naturaloligoribo- or oligodeoxyribonucleotide as well as analogs (e.g.thio-modified phosphodiester or phosphotriester backbone) or employingoligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et al.,Science, 254, 1497 (1991)) which render the base sequence lesssusceptible to enzymatic degradation and hence increases overallstability of the solid support-bound capture base sequence.

Alternatively, a target detection site can be directly linked to a solidsupport via a reversible or irreversible bond between an appropriatefunctionality (L′) on the target nucleic acid molecule (T) and anappropriate functionality (L) on the capture molecule (FIG. 1B). Areversible linkage can be such that it is cleaved under the conditionsof mass spectrometry (i.e., a photocleavable bond such as a chargetransfer complex or a labile bond being formed between relatively stableorganic radicals). Furthermore, the linkage can be formed with L′ beinga quaternary ammonium group, in which case, preferably, the surface ofthe solid support carries negative charges which repel the negativelycharged nucleic acid backbone and thus facilitate the desorptionrequired for analysis by a mass spectrometer. Desorption can occureither by the heat created by the laser pulse and/or, depending on L′,by specific absorption of laser energy which is in resonance with the L′chromophore.

By way of example, the L-L′ chemistry can be of a type of disulfide bond(chemically cleavable, for example, by mercaptoethanol ordithioerythritol), a biotin/streptavidin system, a heterobifunctionalderivative of a trityl ether group (Gildea et al., “A VersatileAcid-Labile Linker for Modification of Synthetic Biomolecules,”Tetrahedron Letters 31: 7095 (1990)) which can be cleaved under mildlyacidic conditions as well as under conditions of mass spectrometry, alevulinyl group cleavable under almost neutral conditions with ahydrazinium/acetate buffer, an arginine-arginine or lysine-lysine bondcleavable by an endopeptidase enzyme like trypsin or a pyrophosphatebond cleavable by a pyrophosphatase, or a ribonucleotide bond in betweenthe oligodeoxynucleotide sequence, which can be cleaved, for example, bya ribonuclease or alkali.

The functionalities, L and L′, can also form a charge transfer complexand thereby form the temporary L-L′ linkage. Since in many cases the“charge-transfer band” can be determined by UV/vis spectrometry (seee.g. Organic Charge Transfer Complex by R. Foster, Academic Press,1969), the laser energy can be tuned to the corresponding energy of thecharge-transfer wavelength and, thus, a specific desorption off thesolid support can be initiated. Those skilled in the art will recognizethat several combinations can serve this purpose and that the donorfunctionality can be either on the solid support or coupled to thenucleic acid molecule to be detected or vice versa.

In yet another approach, a reversible L-L′ linkage can be generated byhomolytically forming relatively stable radicals. Under the influence ofthe laser pulse, desorption (as discussed above) as well as ionizationwill take place at the radical position. Those skilled in the art willrecognize that other organic radicals can be selected and that, inrelation to the dissociation energies needed to homolytically cleave thebond between them, a corresponding laser wavelength can be selected (seee.g., Reactive Molecules by C. Wentrup, John Wiley & Sons, 1984).

An anchoring function L′ can also be incorporated into a targetcapturing sequence (TCS) by using appropriate primers during anamplification procedure, such as PCR (FIG. 4), LCR (FIG. 5) ortranscription amplification (FIG. 6A).

Prior to mass spectrometric analysis, it may be useful to “condition”nucleic acid molecules, for example to decrease the laser energyrequired for volatization and/or to minimize fragmentation. Conditioningis preferably performed while a target detection site is immobilized. Anexample of conditioning is modification of the phosphodiester backboneof the nucleic acid molecule (e.g. cation exchange), which can be usefulfor eliminating peak broadening due to a heterogeneity in the cationsbound per nucleotide unit. Contacting a nucleic acid molecule with analkylating agent such as alkyliodide, iodoacetamide, β-iodoethanol,2,3-epoxy-1-propanol, the monothio phosphodiester bonds of a nucleicacid molecule can be transformed into a phosphotriester bond. Likewise,phosphodiester bonds may be transformed to uncharged derivativesemploying trialkylsilyl chlorides. Further conditioning involvesincorporating nucleotides which reduce sensitivity for depurination(fragmentation during MS) such as N7- or N9-deazapurine nucleotides, orRNA building blocks or using oligonucleotide triesters or incorporatingphosphorothioate functions which are alkylated or employingoligonucleotide mimetics such as PNA.

For certain applications, it may be useful to simultaneously detect morethan one (mutated) loci on a particular captured nucleic acid fragment(on one spot of an array) or it may be useful to perform parallelprocessing by using oligonucleotide or oligonucleotide mimetic arrays onvarious solid supports. “Multiplexing” can be achieved by severaldifferent methodologies. For example, several mutations can besimultaneously detected on one target sequence by employingcorresponding detector (probe) molecules (e.g. oligonucleotides oroligonucleotide mimetics). However, the molecular weight differencesbetween the detector oligonucleotides D1, D2 and D3 must be large enoughso that simultaneous detection (multiplexing) is possible. This can beachieved either by the sequence itself (composition or length) or by theintroduction of mass-modifying functionalities M1-M3 into the detectoroligonucleotide. (FIG. 2)

Mass modifying moieties can be attached, for instance, to either the5′-end of the oligonucleotide (M¹), to the nucleobase (or bases) (M²,M⁷), to the phosphate backbone (M³), and to the 2′-position of thenucleoside (nucleosides) (M⁴, M⁶) or/and to the terminal 3′-position(M⁵). Examples of mass modifying moieties include, for example, ahalogen, an azido, or of the type, XR, wherein X is a linking group andR is a mass-modifying functionality. The mass-modifying functionalitycan thus be used to introduce defined mass increments into theoligonucleotide molecule.

Here the mass-modifying moiety, M, can be attached either to thenucleobase, M² (in case of the c⁷-deazanucleosides also to C-7, M⁷), tothe triphosphate group at the alpha phosphate, M³, or to the 2′-positionof the sugar ring of the nucleoside triphosphate, M⁴ and M6.Furthermore, the mass-modifying functionality can be added so as toaffect chain termination, such as by attaching it to the 3′-position ofthe sugar ring in the nucleoside triphosphate, M⁵. For those skilled inthe art, it is clear that many combinations can serve the purpose of theinvention equally well. In the same way, those skilled in the art willrecognize that chain-elongating nucleoside triphosphates can also bemass-modified in a similar fashion with numerous variations andcombinations in functionality and attachment positions.

Without limiting the scope of the invention, the mass-modification, M,can be introduced for X in XR as well as using oligo-/-polyethyleneglycol derivatives for R. The mass-modifying increment in this case is44, i.e. five different mass-modified species can be generated by justchanging m from 0 to 4 thus adding mass units of 45 (m=0), 89 (m=1), 133(m=2), 177 (m=3) and 221 (m=4) to the nucleic acid molecule (e.g.detector oligonucleotide (D) or the nucleoside triphosphates (FIGS. 3and 6(C), respectively). The oligo/polyethylene glycols can also bemonoalkylated by a lower alkyl such as methyl, ethyl, propyl, isopropyl,t-butyl and the like. A selection of linking functionalities, X, arealso illustrated. Other chemistries can be used in the mass-modifiedcompounds, as for example, those described recently in Oligonucleotidesand Analogues, A Practical Approach (F. Eckstein, editor, IRL Press,Oxford, 1991).

In yet another embodiment, various mass-modifying functionalities, R,other than oligo/polyethylene glycols, can be selected and attached viaappropriate linking chemistries, X. A simple mass-modification can beachieved by substituting H for halogens like F, Cl, Br and/or I, orpseudohalogens such as SCN, NCS, or by using different alkyl, aryl oraralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl,hexyl, phenyl, substituted phenyl, benzyl, or functional groups such asCH₂F, CHF₂, CF₃, Si(CH₃)₃, Si(CH₃)₂(C₂H₅), Si(CH₃)(C₂H₅)₂, Si(C₂H₅)₃.Yet another mass-modification can be obtained by attaching homo- orheteropeptides through the nucleic acid molecule (e.g. detector (D)) ornucleoside triphosphates. One example useful in generating mass-modifiedspecies with a mass increment of 57 is the attachment of oligoglycines,e.g. mass-modifications of 74 (r=1, m=0), 131 (r=1, m=2), 188 (r=1,m=3), 245 (r=1, m=4) are achieved. Simple oligoamides also can be used,e.g., mass-modifications of 74 (r=1, m=0), 88 (r=2, m=0), 102 (r=3,m=0), 116 (r=4, m=0), etc. are obtainable. For those skilled in the art,it will be obvious that there are numerous possibilities in addition tothose mentioned above.

As used herein, the superscript 0-i designates i+1 mass differentiatednucleotides, primers or tags. In some instances, the superscript 0 candesignate an unmodified species of a particular reactant, and thesuperscript i can designate the i-th mass-modified species of thatreactant. If, for example, more than one species of nucleic acids are tobe concurrently detected, then i+1 different mass-modified detectoroligonucleotides (D⁰, D¹, . . . D^(i)) can be used to distinguish eachspecies of mass modified detector oligonucleotides (D) from the othersby mass spectrometry.

Different mass-modified detector oligonucleotides can be used tosimultaneously detect all possible variants/mutants simultaneously (FIG.6B). Alternatively, all four base permutations at the site of a mutationcan be detected by designing and positioning a detector oligonucleotide,so that it serves as a primer for a DNA/RNA polymerase (FIG. 6C). Forexample, mass modifications also can be incorporated during theamplification process.

FIG. 3 shows a different multiplex detection format, in whichdifferentiation is accomplished by employing different specific capturesequences which are position-specifically immobilized on a flat surface(e.g. a ‘chip array’). If different target sequences Ti-Tn are present,their target capture sites TCS1-TCSn will specifically interact withcomplementary immobilized capture sequences C1-Cn. Detection is achievedby employing appropriately mass differentiated detector oligonucleotidesD1-Dn, which are mass differentiated either by their sequences or bymass modifying functionalities M1-Mn.

Preferred mass spectrometer formats for use in the invention are matrixassisted laser desorption ionization (MALDI), electrospray (ES), ioncyclotron resonance (ICR) and Fourier Transform. For ES, the samples,dissolved in water or in a volatile buffer, are injected eithercontinuously or discontinuously into an atmospheric pressure ionizationinterface (API) and then mass analyzed by a quadrupole. The generationof multiple ion peaks which can be obtained using ES mass spectrometrycan increase the accuracy of the mass determination. Even more detailedinformation on the specific structure can be obtained using an MS/MSquadrupole configuration.

In MALDI mass spectrometry, various mass analyzers can be used, e.g.,magnetic sector/magnetic deflection instruments in single or triplequadrupole mode (MS/MS), Fourier transform and time-of-flight (TOF)configurations as is known in the art of mass spectrometry. For thedesorption/ionization process, numerous matrix/laser combinations can beused. Ion-trap and reflectron configurations can also be employed.

The mass spectrometric processes described above can be used, forexample, to diagnose any of the more than 3000 genetic diseasescurrently known (e.g. hemophilias, thalassemias, Duchenne MuscularDystrophy (DMD), Huntington's Disease (HD), Alzheimer's Disease andCystic Fibrosis (CF)) or to be identified.

The following Example 3 provides a mass spectrometer method fordetecting a mutation (ΔF508) of the cystic fibrosis transmembraneconductance regulator gene (CFTR), which differs by only three basepairs (900 daltons) from the wild type of CFTR gene. As describedfurther in Example 3, the detection is based on a single-tube,competitive oligonucleotide single base extension (COSBE) reaction usinga pair of primers with the 3′-terminal base complementary to either thenormal or mutant allele. Upon hybridization and addition of a polymeraseand the nucleoside triphosphate one base downstream, only those primersproperly annealed (i.e., no 3′-terminal mismatch) are extended; productsare resolved by molecular weight shifts as determined by matrix assistedlaser desorption ionization time-of-flight mass spectrometry. For thecystic fibrosis ΔF508 polymorphism, 28-mer ‘normal’ (N) and 30-mer‘mutant’ (M) primers generate 29- and 31-mers for N and M homozygotes,respectively, and both for heterozygotes. Since primer and productmolecular weights are relatively low (<10 kDa) and the mass differencebetween these are at least that of a single ˜300 Da nucleotide unit, lowresolution instrumentation is suitable for such measurements.

In addition to mutated genes, which result in genetic disease, certainbirth defects are the result of chromosomal abnormalities such asTrisomy 21 (Down's syndrome), Trisomy 13 (Platau Syndrome), Trisomy 18(Edward's Syndrome), Monosomy X (Turner's Syndrome) and other sexchromosome aneuploidies such as Klienfelter's Syndrome (XXY).

Further, there is growing evidence that certain DNA sequences maypredispose an individual to any of a number of diseases such asdiabetes, arteriosclerosis, obesity, various autoimmune diseases andcancer (e.g. colorectal, breast, ovarian, lung); chromosomal abnormality(either prenatally or postnatally); or a predisposition to a disease orcondition (e.g. obesity, atherosclerosis, cancer). Also, the detectionof “DNA fingerprints”, e.g. polymorphisms, such as “microsatellitesequences”, are useful for determining identity or heredity (e.g.paternity or maternity).

The following Example 4 provides a mass spectrometer method foridentifying any of the three different isoforms of human apolipoproteinE, which are coded by the E2, E3 and E4 alleles. Here the molecularweights of DNA fragments obtained after restriction with appropriaterestriction endonucleases can be used to detect the presence of amutation.

Depending on the biological sample, the diagnosis for a genetic disease,chromosomal aneuploidy or genetic predisposition can be preformed eitherpre- or post-natally.

Viruses, bacteria, fungi and other organisms contain distinct nucleicacid sequences, which are different from the sequences contained in thehost cell. Detecting or quantitating nucleic acid sequences that arespecific to the infectious organism is important for diagnosing ormonitoring infection. Examples of disease causing viruses that infecthumans and animals and which may be detected by the disclosed processesinclude: Retroviridae (e.g., human immunodeficiency viruses, such asHIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, see Ratner, L.et al., Nature 313: 227-284 (1985); and Wain Hobson, S., et al, Cell 40:9-17 (1985)); HIV-2 (see Guyader et al., Nature 328: 662-669 (1987);European Pat. Publication No. 0 269 520; Chakraborti et al., Nature 328:543-547 (1987); and European Patent Application No. 0 655 501); andother isolates, such as HIV-LP (International Publication No. WO94/00562 entitled “A Novel Human Immunodeficiency Virus”; Picornaviridae(e.g., polio viruses, hepatitis A virus, (Gust, I. D., et al.,Intervirology, Vol. 20, pp. 1-7 (1983); entero viruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains thatcause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,rubella viruses); Flaviridae (e.g., dengue viruses, encephalitisviruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes viruses′); Poxviridae (variola viruses, vaccinia viruses,pox viruses); and Iridoviridae (e.g., African swine fever virus); andunclassified viruses (e.g., the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacterta sps (e.g. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans,Histoplasma capsulatum, Coccidioides immitis, Biastomyces dermatitidis,Chlamydia trachomatis, Candida albicans. Other infectious organisms(i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

The following Example 5 provides a nested PCR and mass spectrometerbased method that was used to detect hepatitis B virus (HBV) DNA inblood samples. Similarly, other blood-borne viruses (e.g., HIV-1, HIV-2,hepatitis C virus (HCV), hepatitis A virus (HAV) and other hepatitisviruses (e.g., non-A-non-B hepatitis, hepatitis G, hepatitis E),cytomegalovirus, and herpes simplex virus (HSV)) can be detected eachalone or in combination based on the methods described herein.

Since the sequence of about 16 nucleotides is specific on statisticalgrounds (even for a genome as large as the human genome), relativelyshort nucleic acid sequences can be used to detect normal and defectivegenes in higher organisms and to detect infectious microorganisms (e.g.bacteria, fungi, protists and yeast) and viruses. DNA sequences can evenserve as a fingerprint for detection of different individuals within thesame species. (Thompson, J. S. and M. W. Thompson, eds., Genetics inMedicine, W. B. Saunders Co., Philadelphia, Pa. (1986).

One process for detecting a wildtype (D^(wt)) and/or a mutant (D^(mut))sequence in a target (T) nucleic acid molecule is shown in FIG. 1C. Aspecific capture sequence (C) is attached to a solid support (ss) via aspacer (S). In addition, the capture sequence is chosen to specificallyinteract with a complementary sequence on the target sequence (T), thetarget capture site (TCS) to be detected through hybridization. However,if the target detection site (TDS) includes a mutation, X, whichincreases or decreases the molecular weight, mutated TDS can bedistinguished from wildtype by mass spectrometry. For example, in thecase of an adenine base (dA) insertion, the difference in molecularweights between D^(wt) and D^(mut) would be about 314 daltons.

Preferably, the detector nucleic acid (D) is designed such that themutation would be in the middle of the molecule and the flanking regionsare short enough so that a stable hybrid would not be formed if thewildtype detector oligonucleotide (D^(wt)) is contacted with the mutatedtarget detector sequence as a control. The mutation can also be detectedif the mutated detector oligonucleotide (D^(mut)) with the matching baseat the mutated position is used for hybridization. If a nucleic acidobtained from a biological sample is heterozygous for the particularsequence (i.e. contain both D^(wt) and D^(mut)), both D^(wt) and D^(mut)will be bound to the appropriate strand and the mass difference allowsby D^(wt) and D^(mut) to be detected simultaneously.

The process of this invention makes use of the known sequenceinformation of the target sequence and known mutation sites, althoughnew mutations can also be detected. For example, as shown in FIG. 8,transcription of a nucleic acid molecule obtained from a biologicalsample can be specifically digested using one or more nucleases and thefragments captured on a solid support carrying the correspondingcomplementary nucleic acid sequences. Detection of hybridization and themolecular weights of the captured target sequences provide informationon whether and where in a gene a mutation is present. Alternatively, DNAcan be cleaved by one or more specific endonucleases to form a mixtureof fragments. Comparison of the molecular weights between wildtype andmutant fragment mixtures results in mutation detection.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications (including international patentapplication Publication Number WO 94/16101 and U.S. Pat. No. 5,605,798,entitled DNA Sequencing by Mass Spectrometry by H. Köster; andinternational patent application Publication Number WO 94/21822 and U.S.Pat. No. 5,622,824, entitled “DNA Sequencing by Mass Spectrometry ViaExonuclease Degradation” by H. Köster), and co-pending patentapplications, (including U.S. patent application Ser. No. 08/406,199,now U.S. Pat. No. 5,605,798 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster), as cited throughout this application arehereby expressly incorporated by reference.

EXAMPLE 1 MALDI-TOF desorption of oligonucleotides Directly on SolidSupports

1 g CPC (Controlled Pore Glass) was functionalized with3-(triethoxysilyl)-epoxypropan to form OH-groups on the polymer surface.A standard oligonucleotide synthesis with 13 mg of the OH-CPG on a DNAsynthesizer (Milligen, Model 7500) employingβ-cyanoethyl-phosphoamidites (Sinha et al., Nucleic Acids Res. 12: 4539(1984)) and TAC N-protecting groups (Köster et al., Tetrahedron 37: 362(1981)) was performed to synthesize a 3′-T₅-50-mer oligonucleotidesequence in which 50 nucleotides are complementary to a “hypothetical”50-mer sequence. T₅ serves as a spacer. Deprotection with saturatedammonia in methanol at room temperature for 2 hours furnished accordingto the determination of the DMT group CPG which contained about 10 μmol55-mer/g CPG. This 55-mer served as a template for hybridizations with a26-mer (with 5′-DMT group) and a 40-mer (without DMT group). Thereaction volume is 100 μl and contains about 1 nmol CPG bound 55-mer astemplate, an equimolar amount of oligonucleotide in solution (26-mer or40-mer) in 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 25 mM NaCl. Themixture was heated for 10′ at 65° C. and cooled at 37° C. during 30′(annealing). The oligonucleotide which has not been hybridized to thepolymer-bound template were removed by centrifugation and threesubsequent washing/centrifugation steps with 100 μl each of ice-cold 50mM ammonium citrate. The beads were air-dried and mixed with matrixsolution (3-hydroxypicolinic acid/10 mM ammonium citrate inacetonitrile/water, 1:1), and analyzed by MALDI-TOF mass spectrometry.The results are presented in FIGS. 10A-10C and 11.

EXAMPLE 2 Electrospray (ES) Desorption and Differentiation of an 18-merand 19-mer

DNA fragments at a concentration of 50 pmole/μl in 2-propanol/10 mMammonium carbonate (1/9, v/v) were analyzed simultaneously by anelectrospray mass spectrometer.

The successful desorption and differentiation of an 18-mer and 19-mer byelectrospray mass spectrometry is shown in FIGS. 12A-12C.

EXAMPLE 3 Detection of the Cystic Fibrosis Mutation, ΔF508, by SingleStep Dideoxy Extension and Analysis by MALDI-TOF Mass SpectrometryMaterial and Methods

PCR Amplification and Strand Immobilization

Amplification was carried out with exon 10 specific primers usingstandard PCR conditions (30 cycles: 1′@95° C., 1′@55° C., 2′@72° C.);the reverse primer was 5′ labelled with biotin and column purified(Oligopurification Cartridge, Cruachem). After amplification the PCRproducts were purified by column separation (Qiagen Quickspin) andimmobilized on streptavidin coated magnetic beads (DynaBeads, Dynal,Norway) according to their standard protocol; DNA was denatured using0.1M NaOH and washed with 0.1M NaOH, 1×B+W buffer and TE buffer toremove the non-biotinylated sense strand.

COSBE Conditions

The beads containing ligated antisense strand were resuspended in 18 μlof Reaction mix (2 μ10×Taq buffer, 1 μL (1 unit) Taq Polymerase, 2 μL of2 mM dGTP, and 13 μL H₂O) and incubated at 80° C. for 5′ before theaddition of Reaction mix 2 (100 ng each of COSBE primers). Thetemperature was reduced to 60° C. and the mixtures incubated for 5′annealing/extension period; the beads were then washed in 25 mMtriethylammonium acetate (TEAA) followed by 50 mM ammonium citrate.

Primer Sequences

All primers were synthesized on a Perseptive Biosystems Expedite 8900DNA Synthesizer using conventional phosphoramidite chemistry (Sinha etal. (1 984) Nucleic Acids Res. 12: 4539. COSBE primers (both containingan intentional mismatch one base before the 3′-terminus) were those usedin a previous ARMS study (Ferrie et al., (1992) Am J Hum Genet 51:251-262) with the exception that two bases were removed from the 5′-endof the normal:

Ex 10 PCR (Forward): 5′-BIO-GCA AGT GAA TCC TGA GCG TG-3′  (SEQ.ID.No.1)

 Ex 10 PCR (Reverse): 5′-GTG TGA AGG GTT CAT ATG C-3′  (SEQ.ID.No. 2)

COSBE ΔF508-N 5′-ATC TAT ATT CAT CAT AGG AAA CAC CAC A-3′ (28-mer)  (SEQ.ID.No. 3)

COSBE ΔF508-M 5′-GTA TCT ATA TTC ATC ATA GGA AAC ACC ATT-3′(30-mer)  (SEQ.ID.No. 4).

Mass Spectrometry

After washing, beads were resuspended in 1 μl 18 Mohm/cm H₂O. 300 nLeach of matrix (Wu et al. (1993) Rapid Commun Mass Spectrom 7: 142-146)solution (0.7 M 3-hydroxypicolinic acid, 0.7 M dibasic ammonium citratein 1:1 H₂O: CH₃CN) and resuspended beads (Tang et al. (1995) RapidCommun Mass Spectrom 8: 727-730) were mixed on a sample target andallowed to air dry. Up to 20 samples were spotted on a probe target diskfor introduction into the source region of an unmodified ThermoBioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operated inreflectron mode with 5 and 20 kV on the target and conversion dynode,respectively. Theoretical average molecular weights (M_(r)(calc)) werecalculated from atomic compositions. Vendor-provided software was usedto determine peak centroids using external calibration; 1.08 Da has beensubtracted from these to correct for the charge-carrying proton mass toyield the test Mr(exp) values.

Scheme

Upon annealing to the bound template, the N and M primers (8508.6 and9148.0 Da, respectively) are presented with dGTP; only primers withproper Watson-Crick base paring at the variable (V) position areextended by the polymerase. Thus if V pairs with 3′-terminal base of N,N is extended to a 8837.9 Da product (N+1). Likewise, if V is properlymatched to the M terminus, M is extended to a 9477.3 Da M+1 product.

Results

FIGS. 14-18 show the representative mass spectra of COSBE reactionproducts. Better results were obtained when PCR products were purifiedbefore the biotinylated anti-sense strand was bound

EXAMPLE 4 Differentiation of Human Apolipoprotein E Isoforms by MassSpectrometry

Apolipoprotein E (Apo E), a protein component of lipoproteins, plays anessential role in lipid metabolism. For example, it is involved withcholesterol transport, metabolism of lipoprotein particles,immunoregulation and activation of a number of lipolytic enzymes.

There are common isoforms of human Apo E (coded by E2, E3, and E4alleles). The most common is the E3 allele. The E2 allele has been shownto decrease the cholesterol level in plasma and therefore may have aprotective effect against the development of atherosclerosis. Finally,the E4 isoform has been correlated with increased levels of cholesterol,conferring predisposition to atherosclerosis. Therefore, the identity ofthe apo E allele of a particular individual is an important determinantof risk for the development of cardiovascular disease.

As shown in FIG. 19, a sample of DNA encoding apolipoprotein E can beobtained from a subject, amplified (e.g. via PCR); and the PCR productcan be digested using an appropriate enzyme (e.g. Cfol). The restrictiondigest obtained can then be analyzed by a variety of means. As shown inFIGS. 20A-20B, the three isotypes of apolipoprotein E (E2, E3 and E4have different nucleic acid sequences and therefore also havedistinguishable molecular weight values.

As shown in FIGS. 21A-21C, different Apolipoprotein E genotypes exhibitdifferent restriction patterns in a 3.5% MetPhor Agarose Gel or 12%polyacrylamide gel. As shown in FIGS. 22A-22B and 23A-23B, the variousapolipoprotein E genotypes can also be accurately and rapidly determinedby mass spectrometry.

EXAMPLE 5 Detection of Hepatitis B Virus in Serum Samples Materials andMethod

Sample Preparation

Phenol/chloroform extraction of viral DNA and the final ethanolprecipitation was done according to standard protocols.

First PCR

Each reaction was performed with 5 μl of the DNA preparation from serum.15 pmol of each primer and 2 units Taq DNA polymerase (Perkin Elmer,Weiterstadt, Germany) were used. The final concentration of each dNTPwas 200 μM, and the final volume of the reaction was 50 μl. 10x PCRbuffer (Perkin Elmer, Weiterstadt, Germany) contained 100 mM Tris-HCl,pH 8.3, 500 mM KCl, 15 mM MgCl₂, and 0.01% gelatine (w/v).

Primer sequences:

Primer 1:5′-GCTTTGGGGCATGGACATTGACCCGTATAA-3′  (SEQ ID NO 5)

Primer 2:5′-CTGACTACTAATTCCCTGGATGCTGGGTCT-3′  (SEQ ID NO 6)

Nested PCR

Each reaction was performed either with 1 μl of the first reaction orwith a 1:10 dilution of the first PCR as template, respectively. 100pmol of each primer, 2.5 u Pfu(exo−) DNA polymerase (Stratagene,Heidelberg, Germany), a final concentration of 200 μM of each dNTPs and5 μl 10x Pfu buffer (200 mM Tris-HCl, pH 8.75, 100 mM KCl, 100 mM(NH₄)₂SO₄, 20 mM MgSO_(4,) 1% Triton X-100, and 1mg/ml BSA, (Stratagene,Heidelberg, Germany) were used in a final volume of 50 μl. The reactionswere performed in a thermocycler (OmniGene, MWG-Biotech, Ebersberg,Germany) using the following program: 92° C. for 1 minute, 60° C. for 1minute and 72° C. for 1 minute with 20 cycles. The sequences of the HBVoligodeoxynucleotides (purchased HPLC-purified at MWG-Biotech,Ebersberg, Germany) are provided below.

HBV 13: 5′-TTGCCTGAGTGCAGTATGGT-3′  (SEQ.ID.No. 7)

HBV 15bio: Biotin-5′-AGCTCTATATCGGGAAGCCCT-3′  (SEQ.ID.No. 8)

Purification of PCR Products

For the recording of each spectrum, one PCR, 50 μl (performed asdescribed above) was used. Purification was done according to thefollowing procedure: Ultrafiltration was done using Ultrafree-MCfiltration units (Millipore, Eschborn, Germany) according to theprotocol of the provider with centrifugation at 8000 rpm for 20 minutes.25 μl (10 μg/μl) streptavidin Dynabeads (Dynal, Hamburg, Germany) wereprepared according to the instructions of the manufacturer andresuspended in 25 μl of B/W buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 2M NaCl). This suspension was added to the PCR samples still in thefiltration unit and the mixture was incubated with gentle shaking for 15minutes at ambient temperature. The suspension was transferred in a 1.5ml Eppendorf tube and the supernatant was removed with the aid of aMagnetic Particle Collector, MPC, (Dynal, Hamburg, Germany). The beadswere washed twice with 50 μl of 0.7 M ammonium citrate solution, pH 8.0(the supernatant was removed each time using the MPC). Cleavage from thebeads can be accomplished by using formamide at 90° C. The supernatantwas dried in a speedvac for about an hour and resuspended in 4 μl ofultrapure water (MilliQ UF plus Millipore, Eschborn, Germany). Thepreparation was used for MALDI-TOF MS analysis.

MALDI-TOR MS

Half a microliter of the sample was pipetted onto the sample holder,then immediately mixed with 0.5 μl matrix solution (0.7 M3-hydroxy-picolinic acid, 50% acetonitrile, 70 mM ammonium citrate).This mixture was dried at ambient temperature and introduced into themass spectrometer. All spectra were taken in positive ion mode using aFinnigan MAT Vision 2000 (Finnigan MAT, Bremen, Germany), equipped witha reflectron 5 keV ion source, 20 keV postacceleration) and a 337 nmnitrogen laser. Calibration was done with a mixture of a 40-mer and a100-mer. Each sample was measured with different laser energies. In thenegative samples, the PCR product was detected neither with less norwith higher laser energies. In the positive samples the PCR product wasdetected at different places of the sample spot and also with varyinglaser energies.

Results

A nested PCR system was used for the detection of HBV DNA in bloodsamples employing oligonucleotides complementary to the c region of theHBV genome (primer 1: beginning at map position 1763, primer 2 beginningat map position 2032 of the complementary strand) encoding the HBV coreantigen (HBV cAg). DNA was isolated from patients serum according tostandard protocols. A first PCR was performed with the DNA from thesepreparations using a first set of primers. If HBV DNA was present in thesample, a DNA fragment of 269 bp was generated.

In the second reaction, primers which were complementary to a regionwithin the PCR fragment generated in the first PCR were used. IfHBV-related PCR products were present in the first PCR, a DNA fragmentof 67 bp was generated (see FIG. 25A) in this nested PCR. The usage of anested PCR system for detection provides a high sensitivity and alsoserves as a specificity control for the external PCR (Rolfs, A. et al.,PCR: Clinical Diagnostics and Research, Springer, Heidelberg, 1992). Afurther advantage is that the amount of fragments generated in thesecond PCR is high enough to ensure an unproblematic detection althoughpurification losses cannot be avoided.

The samples were purified using ultrafiltration to remove the primersprior to immobilization on streptavidin DynaBeads. This purification wasdone because the shorter primer fragments were immobilized in higheryield on the beads due to steric reasons. The immobilization was donedirectly on the ultrafiltration membrane to avoid substance losses dueto unspecific absorption on the membrane. Following immobilization, thebeads were washed with ammonium citrate to perform cation exchange(Pieles, U. et al., (1992) Nucleic Acids Res 21: 3191-3196). Theimmobilized DNA was cleaved from the beads using 25% ammonia whichallows cleavage of DNA from the beads in a very short time, but does notresult in an introduction of sodium cations.

The nested PCRs and the MALDI TOF analysis were performed withoutknowing the results of serological analysis. Due to the unknown virustiter, each sample of the first PCR was used undiluted as template andin a 1:10 dilution, respectively.

Sample 1 was collected from a patient with chronic active HBV infection,was positive in HBs and HBe-antigen tests, but negative in a dot blotanalysis. Sample 2 was a serum sample from a patient with an active HBVinfection and a massive viremia who was HBV positive in a dot blotanalysis. Sample 3 was a denatured serum sample, therefore noserological analysis could be performed but an increased level oftransaminases indicating liver disease was detected. In autoradiographanalysis (FIG. 24), the first PCR of this sample was negative.Nevertheless, there was some evidence of HBV infection. This sample isof interest for MALDI-TOF analysis, because it demonstrates that evenlow-level amounts of PCR products can be detected after the purificationprocedure. Sample 4 was from a patient who was cured of HBV infection.Samples 5 and 6 were collected from patients with a chronic active HBVinfection.

FIG. 24 shows the results of a PAGE analysis of the nested PCR reaction.A PCR product is clearly revealed in samples 1, 2, 5 and 6. In sample 4no PCR product was generated; it is indeed HBV negative, according tothe serological analysis. Amplification artifacts are visible in lanes2, 5 and 6, if non-diluted template was used. These artifacts were notgenerated if the template was used in a 1:10 dilution. In sample 3, PCRproduct was only detectable if the template was not diluted. The resultsof PAGE analysis are in agreement with the data obtained by serologicalanalysis except for sample 3 as discussed above.

FIG. 25A shows a mass spectrum of a nested PCR product from samplenumber 1 generated and purified as described above. The signal at 20754Da represents the single stranded PCR product (calculated: 20735 Da, asthe average mass of both strands of the PCR product cleaved from thebeads). The mass difference of calculated and obtained mass is 19 Da(0.09%). As shown in FIG. 25A, sample number 1 generated a high amountof PCR product, resulting in an unambiguous detection.

FIG. 25B shows a spectrum obtained from sample number 3. As depicted inFIG. 24, the amount of PCR product generated in this section issignificantly lower than that from sample number 1. Nevertheless, thePCR product is clearly revealed with a mass of 20751 Da (calculated20735). The mass difference is 16 Da (0.08%). The spectrum depicted inFIG. 25C was obtained from sample number 4 which is HBV negative (as isalso shown in FIG. 24). As expected no signals corresponding to the PCRproduct could be detected. All samples shown in FIGS. 25A-25C wereanalyzed with MALDI-TOF MS, whereby PCR product was detected in all HBVpositive samples, but not in the HBV negative samples. These resultswere reproduced in several independent experiments.

EXAMPLE 6 Analysis of Ligase Chain Reaction Products Via MALDI-TOF MassSpectrometry Materials and Methods

Oligodeoxynucleotides

Except for the biotinylated oligonucleotide, all other oligonucleotideswere synthesized in a 0.2 μmol scale on a MilliGen 7500 DNA Synthesizer(Millipore, Bedford, Mass., USA) using the β-cyanoethylphosphoamiditemethod (Sinha, N. D. et al., (1984) Nucleic Acids Res. 12: 4539-4577).The oligodeoxynucleotides were RP-HPLC-purified and deprotectedaccording to standard protocols. The biotinylated oligodeoxynucleotidewas purchased (HPLC-purified) from Biometra, Gottingen, Germany).

Sequences and calculated masses of the oligonucleotides used

Oligodeoxynucleotide A: 5′-p-TTGTGCCACGCGGTTGGGAATGTA-3′ (7521Da)  (SEQ.ID.No. 9)

Oligodeoxynucleotide B: 5′-p-AGCAACGACTGTTTGCCCGCCAGTTG-3′ (7948Da)  (SEQ.ID.No 10)

 Oigodeoxynucleotide C: 5′-bio-TACATTCCCAACCGCGTGGCACAAC-3′ (7960Da)  (SEQ.ID.No. 11)

Oligodeoxynucleotide D: 5′-p-AACTGGCGGGCAAACAGTCGTTGCT-3′ (7708Da)  (SEQ.ID.No. 12)

5′-Phosphorylation of Oligonucleotides A and D

This was performed with polynucleotide Kinase (Boehringer, Mannheim,Germany) according to published procedures; the 5′-phosphorylatedoligonucleotides were used unpurified for LCR.

Ligase Chain Reaction

The LCR was performed with Pfu DNA ligase and a ligase chain reactionkit (Stratagene, Heidelberg, Germany) containing two differentpBluescript KII phagemids, one carrying the wildtype form of the E. colilacl gene and the other one a mutant of the gene with a single pointmutation of bp 191 of the lacl gene.

The following LCR conditions were used for each reaction: 100 pgtemplate DNA (0.74 fmol) with 500 pg sonified salmon sperm DNA ascarrier, 25 ng (3.3 pmol) of each 5′-phosphorylated oligonucleotide, 20ng (2.5 pmol) of each non-phosphorylated oligonucleotide, and 4 u PfuDNA ligase in a final volume of 20 μl buffered by Pfu DNA ligasereaction buffer (Stratagene, Heidelberg, Germany). In a modelexperiment, a chemically synthesized ss 50-mer was used (1 fmol) astemplate, and in this case oligo C was also biotinylated. All reactionswere performed in a thermocycler (OmniGene, MWG-Biotech, Ebersberg,Germany) with the following program: 4 minutes 92° C., 2 minutes 60° C.and 25 cycles of 20 seconds 92° C., 40 seconds 60° C. Except for HPLCanalysis, the biotinylated ligation educt C was used. In a controlexperiment the biotinylated and non-biotinylated oligonucleotidesrevealed the same gel electrophoretic results. The reactions wereanalyzed on 7.5% polyacrylamide gels. Ligation product 1 (oligo A and B)calculated mass: 15450 Da; ligation product 2 (oligo C and D) calculatedmass: 15387 Da.

SMART-HPLC

Ion exchange HPLC (IE HPLC) was performed on the SMART-system(Pharmacia, Freibrug, Germany) using a Pharmacia Mono Q, PC 1.6/5column. Eluents were buffer A (25 mM Tris-HCl, 1 mM EDTA and 0.3 M NaClat pH 8.0) and buffer B (same as A, but 1 M NaCl). Starting with 100% Afor 5 minutes at a flow rate of 50 μl/min a gradient was applied from 0to 70% B in 30 minutes, then increased to 100% B in 2 minutes and heldat 100% B for 5 minutes. Two pooled LCR volumes (40 μl ) performed witheither wildtype or mutant template were injected.

Sample Preparation for MALDI-TOF-MS

Preparation of immobilized DNA: For the recording of each spectrum twoLCRs (performed as described above) were pooled and diluted 1:1 with 2×B/W buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl). To the samples5 μl streptavidin DynaBeads (Dynal, Hamburg, Germany) were added and themixture was allowed to bind with gentle shaking for 15 minutes atambient temperature. The supernatant was removed using a MagneticParticle Collector, MPC (Dynal, Hamburg, Germany) and the beads werewashed twice with 50 μl of 0.7 M ammonium citrate solution (pH 8.0) (thesupernatant was removed each time using the MPC). The beads wereresuspended in 1 μl of ultrapure water (MilliQ, Millipore, Bedford,Mass., USA). This suspension was directly used for MALDI-TOF-MS analysisas described below.

Combination of ultrafiltration and streptavidin DynaBeads: For therecording of spectrum two LCRs (performed as described above) werepooled, diluted 1:1 with 2× B/W buffer and concentrated with a 5000 NMWLUltrafree-MC filter unit (Millipore, Eschborn, Germany) according to theinstructions of the manufacturer. After concentration, the samples werewashed with 300 μl 1× B/W buffer and 5 μl streptavidin DynaBeads wereadded. The beads were washed once on the Ultrafree-MC filtration unitwith 300 μl of 1× B/W buffer and processed as described above. The beadswere resuspended in 30 to 50 μl of 1× B/W buffer and transferred in a1.5 ml Eppendorf tube. The supernatant was removed and the beads werewashed twice with 50 μl of 0.7 M ammonium citrate (pH 8.0). Finally, thebeads were washed once with 30 μl of acetone and resuspended in 1 μl ofultrapure water. The ligation mixture, after immobilization on thebeads, was used for MALDI-TOF-MS analysis as described below.

MALDI-TOF-MS

A suspension of streptavidin-coated magnetic beads with the immobilizedDNA was pipetted onto the sample holder, then immediately mixed with 0.5μl matrix solution (0.7 M 3-hydroxypicolinic acid in 50% acetonitrile,70 mM ammonium citrate). This mixture was dried at ambient temperatureand introduced into the mass spectrometer. All spectra were taken inpositive ion mode using a Finnigan MAT Vision 2000 (Finnigan MAT,Bremen, Germany), equipped with a reflectron (5 ke V ion source, 20 keVpostacceleration) and a nitrogen laser (337 nm). For the analysis of PfuDNA ligase, 0.5 μl of the solution was mixed on the sample holder with 1μl of matrix solution and prepared as described above. For the analysisof unpurified LCRs, 1 μl of an LCR was mixed with 1 μl matrix solution.

RESULTS AND DISCUSSION

The E. coli lacl gene served as a simple model system to investigate thesuitability of MALDI-TOF-MS as a detection method for products generatedin ligase chain reactions. This template system consists of an E. colilacl wildtype gene in a pBluescript KII phagemid and an E. coli laclgene carrying a single point mutation at bp 191 (C to T transition) inthe same phagemid. Four different oligonucleotides were used, which wereligated only if the E. coli lacI wildtype gene was present (FIG. 26).

LCR conditions were optimized using Pfu DNA ligase to obtain at least 1pmol ligation product in each positive reaction. The ligation reactionswere analyzed by polyacrylamide gel electrophoresis (PAGE) and HPLC onthe SMART system (FIGS. 27, 28 and 29). FIG. 27 shows a PAGE of apositive LCR with wildtype template (lane 1), a negative LCR with mutanttemplate (1 and 2) and a negative control which contains enzyme,oligonucleotides and no template. The gel electrophoresis clearly showsthat the ligation product (50bp) was produced only in the reaction withwildtype template whereas neither the template carrying the pointmutation nor the control reaction with salmon sperm DNA generatedamplification products. In FIG. 28, HPLC was used to analyze two pooledLCRs with wildtype template performed under the same conditions. Theligation product was clearly revealed. FIG. 29 shows the results of aHPLC in which two pooled negative LCRs with mutant template wereanalyzed. These chromatograms confirm the data shown in FIG. 27 and theresults taken together clearly demonstrate, that the system generatesligation products in a significant amount only if the wildtype templateis provided.

Appropriate control runs were performed to determine retention times ofthe different compounds involved in the LCR experiments. These includethe four oligonucleotides (A, B, C, and D), a synthetic ds 50-mer (withthe same sequence as the ligation product), the wildtype template DNA,sonicated salmon sperm DNA and the Pfu DNA ligase in ligation buffer.

In order to test which purification procedure should be used before aLCR reaction can be analyzed by MALDI-TOF-MS, aliquots of an unpurifiedLCR (FIG. 30A) and aliquots of the enzyme stock solution (FIG. 30B) wereanalyzed with MALDI-TOF-MS. It turned out that appropriate samplepreparation is absolutely necessary since all signals in the unpurifiedLCR correspond to signals obtained in the MALDI-TOF-MS analysis of thePfu DNA ligase. The calculated mass values of oligo A and the ligationproduct are 7521 Da and 15450 Da, respectively. The data in FIGS.30A-30B show that the enzyme solution leads to mass signals which dointerfere with the expected signals of the ligation educts and productsand therefore makes an unambiguous signal assignment impossible.Furthermore, the spectra showed signals that the detergent Tween 20 thatis in the enzyme storage buffer influences the crystallization behaviorof the analyte/matrix mixture in an unfavorable way.

In one purification format streptavidin-coated magnetic beads were used.As was shown in a recent paper, the direct desorption of DNA immobilizedby Watson-Crick base pairing to a complementary DNA fragment covalentlybound to the beads is possible and the non-biotinylated strand will bedesorbed exclusively (Tang, K. et al., Nucleic Acids Res. 23: 3126-3131(1995)). This approach in using immobilized ds DNA ensures that only thenon-biotinylated strand will be desorbed. If non-immobilized ds DNA isanalyzed both strands are desorbed (Tang, K. et al., Rapid Comm. MassSpectrom. 7: 183-186 (1994)) leading to broad signals depending on themass difference of the two strands. Therefore, employing this system forLCR, only the non-ligated oligonucleotide A, with a calculated mass of7521 Da, and the ligation product from oligo A and oligo B (calculatedmass: 15450 Da) will be desorbed if oligo C is biotinylated at the 5′end and immobilized on steptavidin-coated beads. This results in asimple and unambiguous identification of the LCR educts and products.

FIG. 31A shows a MALDI-TOF mass spectrum obtained from two pooled LCRs(performed as described above) purified on streptavidin DynaBeads anddesorbed directly from the beads, indicating that the purificationmethod used was efficient (compared with FIGS. 30A-30B). A signal whichrepresents the unligated oligo A and a signal which corresponds to theligation product could be detected. The agreement between the calculatedand the experimentally found mass values is remarkable and allows anunambiguous peak assignment and accurate detection of the ligationproduct. In contrast, no ligation product but only oligo A could bedetected in the spectrum obtained from two pooled LCRs with mutatedtemplate (FIG. 31B). The specificity and selectivity of the LCRconditions and the sensitivity of the MALDI-TOF detection is furtherdemonstrated when performing the ligation reaction in the absence of aspecific template. FIG. 32 shows a spectrum obtained from two pooledLCRs in which only salmon sperm DNA was used as a negative control; onlyoligo A could be detected, as expected.

While the results shown in FIG. 31A can be correlated to lane 1 of thegel in FIG. 27, the spectrum shown in FIG. 31B is equivalent to lane 2in FIG. 27, and finally also the spectrum in FIG. 32 corresponds to lane3 in FIG. 27. The results are in congruence with the HPLC analysispresented in FIGS. 28 and 29. While both gel electrophoresis (FIG. 27)and HPLC (FIGS. 28 and 29) reveal either an excess or almost equalamounts of ligation product over ligation educts, the analysis byMALDI-TOF mass spectrometry produces a smaller signal for the ligationproduct (FIG. 31A).

The lower intensity of the ligation product signal could be due todifferent desorption/ionization efficiencies between 24- and a 50-mer.Since the T_(m) value of a duplex with 50 compared to 24 base pairs issignificantly higher, more 24-mer could be desorbed. A reduction insignal intensity can also result from a higher degree of fragmentationin the case of the longer oligonucleotides.

Regardless of the purification with streptavidin DynaBeads, FIG. 32reveals traces of Tween 20 in the region around 2000 Da. Substances witha viscous consistency negatively influence the process ofcrystallization and therefore can be detrimental to mass spectrometeranalysis. Tween 20 and also glycerol which are part of enzyme storagebuffers therefore should be removed entirely prior to mass spectrometeranalysis. For this reason an improved purification procedure whichincludes an additional ultrafiltration step prior to treatment withDynaBeads was investigated. Indeed, this sample purification resulted ina significant improvement of MALDI-TOF mass spectrometric performance.

FIGS. 33A-33B show spectra obtained from two pooled positive (33A) andnegative (33B) LCRs, respectively. The positive reaction was performedwith a chemically synthesized, single strand 50-mer as template with asequence equivalent to the ligation product of oligo C and D. Oligo Cwas 5′-biotinylated. Therefore the template was not detected. Asexpected, only the ligation product of Oligo A and B (calculated mass15450 Da) could be desorbed from the immobilized and ligated oligo C andD. This newly generated DNA fragment is represented by the mass signalof 15448 Da in FIG. 33A. Compared to FIG. 31A, this spectrum clearlyshows that this method of sample preparation produces signals withimproved resolution and intensity.

EXAMPLE 7 Mutation Detection by Solid Phase Oligo Base Extension of aPrimer and Analysis by MALDI-TOF Mass Spectrometry

Summary

The solid-phase oligo base extension method detects point mutations andsmall deletions as well as small insertions in amplified DNA. The methodis based on the extension of a detection primer that anneals adjacent toa variable nucleotide position on an affinity-captured amplifiedtemplate, using a DNA polymerase, a mixture of three dNTPs, and themissing one dideoxy nucleotide. The resulting products are evaluated andresolved by MALDI-TOF mass spectrometry without further labelingprocedures. The aim of the following experiment was to determine mutantand wildtype alleles in a fast and reliable manner.

Description of the Experiment

The method used a single detection primer followed by an oligonucleotideextension step to give products, differing in length by some basesspecific for mutant or wildtype alleles which can be easily resolved byMALDI-TOF mass spectrometry. The method is described by using as examplethe exon 10 of the CFTR-gene. Exon 10 of this gene leads, in thehomozygous state, to the clinical phenotype of cystic fibrosis.

Materials and Methods

Genomic DNA

Genomic DNA were obtained from healthy individuals, individualshomozygous or heterozygous for the ΔF508 mutation, and one individualheterozygous for the 1506S mutation. The wildtype and mutant alleleswere confirmed by standard Sanger sequencing.

PCR Amplification of Exon 10 of the CFTR Gene

The primers for PCR amplification were CFEx10-F(5′-GCAAGTGAATCCTGAGCGTG-3′ (SEQ.ID.No. 13) located in intron 9 andbiotinylated) and CFEx10-R (5′-GTGTGAAGGGCGTG-3′, (SEQ.ID.No. 14)located in intron 10). Primers were used at a concentration of 8 pmol.Taq-polymerase including 10× buffer was purchased fromBoehringer-Mannheim and dTNPs were obtained from Pharmacia. The totalreaction volume was 50 μl. Cycling conditions for PCR were initially 5min. at 95° C., followed by 1 min. at 94° C., 45 sec at 53° C., and 30sec at 72° C. for 40 cycles with a final extension time of 5 min at 72°C.

Purification of the PCR Products

Amplification products were purified by using Qiagen's PCR purificationkit (No. 28106) according to manufacturer's instructions. The elution ofthe purified products from the column was done in 50 μl TE-buffer (10 mMTris, 1 mM EDTA, pH 7.5).

Affinity-capture and Denaturation of the Double Stranded DNA

10 μl aliquots of the purified PCR product were transferred to one wellof a streptavidin-coated microtiter plate (No. 1645684Boehringer-Mannheim or No. 95029262 Labsystems). Subsequently, 10 μlincubation buffer (80 mM sodium phosphate, 400 mM NaCl, 0.4% Tween 20,pH 7.5) and 30 μl water were added. After incubation for 1 hour at roomtemperature the wells were washed three times with 200 μl washing buffer(40 mM Tris, 1 mM EDTA, 50 mM NaCl, 0.1% Tween 20, pH 8.8). Todenaturate the double stranded DNA, the wells were treated with 100 μlof a 50 mM NaOH solution for 3 min. Hence, the wells were washed threetimes with 200 μl washing buffer.

Oligo Base Extension Reaction

The annealing of 25 pmol detection primer (CF508:5′-CTATATTCATCATAGGAAACACCA-3′ (SEQ ID No. 15)) was performed in 50 μlannealing buffer (20 mM Tris, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO, 1%Triton X-100, pH 8.75) at 50° C. for 10 min. The wells were washed threetimes with 200 μl washing buffer and once in 200 μl TE buffer. Theextension reaction was performed by using some components of the DNAsequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia.The total reaction volume was 45 μl, consisting of 21 μl sample, 6 μlSequenase-buffer, 3 μl 10 mM DTT solution, 4.5 μl of 0.5 mM of threedNTPs, 4.5 μl of 2 mM of the missing one ddNTP, 5.5 μl glycerol enzymedilution buffer, 0.25 μl Sequenase 2.0, and 0.25 μl pyrophosphatase. Thereaction was pipetted on ice and then incubated for 15 min. at roomtemperature and for 5 min. at 37° C. Hence, the wells were washed threetimes with 200 μl washing buffer and once with 60 μl of a 70 mMNH₄-citrate solution.

Denaturation and Precipitation of the Extended Primer

The extended primer was denatured in 50 μl 10% DMSO (dimethylsulfoxide)in water at 80° C. for 10 min. For precipitation, 10 μl NH₄-acetate (pH6.5), 0.5 μl glycogen (10 mg/ml water, Sigma No.

G1765), and 100 μl absolute ethanol were added to the supernatant andincubated for 1 hour at room temperature. After centrifugation at 13,000g for 10 min., the pellet was washed in 70% ethanol and resuspended in 1μl 18 Mohm/cm H₂O water.

Sample Preparation and Analysis on MALDI-TOF Mass Spectrometry

Sample preparation was performed by mixing 0.3 μl each of matrixsolution (0.7 M 3-hydroxypicolinic acid, 0.07 M dibasic ammonium citratein 1:1 H₂O:CH₃CN) and of resuspended DNA/glycogen pellet on a sampletarget and allowed to air dry. Up to 20 samples were spotted on a probetarget disk for introduction into the source region of an unmodifiedThermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operatedin reflectron mode with 5 and 20 kV on the target and conversion dynode,respectively. Theoretical average molecular mass (M_(r)(calc)) werecalculated from atomic compositions; reported experimental Mr(M_(r)(exp)) values are those of the singly-protonated form, determinedusing external calibration.

Results

The aim of the experiment was to develop a fast and reliable methodindependent of exact stringencies for mutation detection that leads tohigh quality and high throughput in the diagnosis of genetic diseases.Therefore a special kind of DNA sequencing (oligo base extension of onemutation detection primer) was combined with the evaluation of theresulting mini-sequencing products by matrix-assisted laser desorptionionization (MALDI) mass spectrometry (MS). The time-of-flight (TOF)reflectron arrangement was chosen as a possible mass measurement system.To prove this hypothesis, the examination was performed with exon 10 ofthe CFTR-gene, in which some mutations could lead to the clinicalphenotype of cystic fibrosis, the most common monogenetic disease in theCaucasian population.

The schematic presentations given in FIGS. 34A-34B show the expectedshort sequencing products with the theoretically calculated molecularmass of the wildtype and various mutations of exon 10 of the CFTR-gene.The short sequencing products were produced using either ddTTP (FIG.34A) or ddCTP (FIG. 34B) to introduce a definitive sequence related stopin the nascent DNA strand. The MALDI-TOF-MS spectra of healthy, mutationheterozygous, and mutation homozygous individuals are presented in FIGS.35A-35B. All samples were confirmed by standard Sanger sequencing whichshowed no discrepancy in comparison to the mass spec analysis. Theaccuracy of the experimental measurements of the various molecularmasses was within a range of minus 21.8 and plus 87.1 dalton (Da) to therange expected. This is a definitive interpretation of the resultsallowed in each case. A further advantage of this procedure is theunambiguous detection of the Δ1507 mutation. In the ddTTP reaction, thewildtype allele would be detected, whereas in the ddCTP reaction thethree base pair deletion would be disclosed.

The method described is highly suitable for the detection of singlepoint mutations or microlesions of DNA. Careful choice of the mutationdetection primers will open the window of multiplexing and lead to ahigh throughput including high quality in genetic diagnosis without anyneed for exact stringencies necessary in comparable allele-specificprocedures. Because of the uniqueness of the genetic information, theoligo base extension of mutation detection primer is applicable in eachdisease gene or polymorphic region in the genome like variable number oftandem repeats (VNTR) or other single nucleotide polymorphisms (e.g.,apolipoprotein E gene).

EXAMPLE 8 Detection of Polymerase Chain Reaction Products Containing7-Deazapurine Moieties with Matrix-Assisted Laser Desorption/IonizationTime-of-Flight (MALDI-TOF) Mass Spectrometry Materials and Methods

PCR Amplifications

The following oligodeoxynucleotide primers were either synthesizedaccording to standard phosphoamidite chemistry (Sinha, N. D. et al.,Tetrahdron Let. 24: 5843-5846 (1983); Sinha, N. D. et al., Nucleic AcidsRes. 12: 4539-4557 (1984)) on a Milligen 7500 DNA synthesizer(Millipore, Bedford, Mass., USA) in 200 nmol scales or purchased fromMWG-Biotech (Ebersberg, Germany, primer 3) and Biometra (Goettingen,Germany, primers 6-7).

primer 1: 5′-GTCACCCTCGACCTGCAG-3′ (SEQ.ID.NO.16); primer 2:5′-TTGTAAAACGACGGCCAGT-3′ (SEQ.ID.NO.17); primer 3:5′-CTTCCACCGCGATGTTGA-3′ (SEQ.ID.NO.18); primer 4:5′-CAGGAAACAGCTATGAC-3′ (SEQ.ID.NO.19); primer 5:5′-GTAAAACGACGGCCAGT-3′ (SEQ.ID.NO.20); primer 6:5′-GTCACCCTCGACCTGCAgC-3′ (SEQ.ID.NO.21); (g: RiboG) primer 7:5′-GTTGTAAAACGAGGGCCAgT-3′ (SEQ.ID.NO.22). (g: RiboG)

The 99-mer and 200-mer DNA strands (modified and unmodified) as well asthe ribo- and 7-deaza-modified 100-mer were amplified from pRFc1 DNA (10ng, generously supplied S. Feyerabend, University of Hamburg) in 100 μlreaction volume containing 10 mmol/L KCl, 10 mmol/L (NH₄)₂SO₄, 20 mmol/LTris HCl (pH=8.8), 2 mmol/L MgSO₄, (exo(−)Pseudococcus furiosus(Pfu)-Buffer, Pharmacia, Freiburg, Germany), 0.2 mmol/L each dNTP(Pharmacia, Freiburg, Germany), 1 μl of each primer and 1 unit ofexo(−)Pfu DNA polymerase (Stratagene, Heidelberg, Germany).

For the 99-mer primers 1 and 2, for the 200-mer primers 1 and 3 and forthe 100-mer primers 6 and 7 were used. To obtain 7-deazapurine modifiednucleic acids, during PCR-amplification dATP and dGTP were replaced with7-deaza-dATP and 7-deaza-dGTP. The reaction was performed in a thermalcycler (OmniGene, MWG-Biotech, Ebersberg, Germany) using the cycle:denaturation at 95° C. for 1 min., annealing at 51° C. for 1 min. andextension at 72° C. for 1 min. For all PCRs the number of reactioncycles was 30. The reaction was allowed to extend for additional 10 min.at 72° C. after the last cycle.

The 103-mer DNA strands (modified and unmodified) were amplified fromM13mp18 RFl DNA (100 ng, Pharmacia, Freiburg, Germany) in 100 μlreaction volume using primers 4 and 5; all other concentrations wereunchanged. The reaction was performed using the cycle: denaturation at95° C. for 1 min., annealing at 40° C. for 1 min., and extension at 72°C. for 1 min. After 30 cycles for the unmodified and 40 cycles for themodified 103-mer respectively, the samples were incubated for anadditional 10 min. at 72° C.

Synthesis of 5′-[³²P]-labeled PCR-primers

Primers 1 and 4 were 5′-[³²P]-labeled employing T4-polynucleotide kinase(Epicentre Technologies) and(γ-³²P)-ATP. (BLU/NGG/502A, Dupont, Germany)according to the protocols of the manufacturer. The reactions wereperformed substituting 10% of primer 1 and 4 in PCR with the labeledprimers under otherwise unchanged reaction conditions. The amplifiedDNAs were separated by gel electrophoresis on a 10% polyacrylamide gel.The appropriate bands were excised and counted on a Packard TRI-CARB460C liquid scintillation system (Packard, Conn., USA).

Primer-cleavage from Ribo-modified PCR-product

The amplified DNA was purified using Ultrafree-MC filter units (30,000NMWL), it was then redissolved in 100 μl of 0.2 mol/L NaOH and heated at95° C. for 25 minutes. The solution was then acidified with HCl (1mol/L) and further purified for MALDI-TOF analysis employingUltrafree-MC filter units (30,000 NMWL) as described below.

Purification of PCR Products

All samples were purified and concentrated using Ultrafree-MC units30,000 NMWL (Millipore, Eschborn, Germany) according to themanufacturer's description. After lyophilization, PCR products wereredissolved in 5 μl (3 μl for the 200-mer) of ultrapure water. Thisanalyte solution was directly used for MALDI-TOF measurements.

MALDI-TOF MS

Aliquots of 0.5 μl of analyte solution and 0.5 μl of matrix solution(0.7 mol/L 3-HPA and 0.07 mol/L ammonium citrate in acetonitrile/water(1:1, v/v) were mixed on a flat metallic sample support. After drying atambient temperature the sample was introduced into the mass spectrometerfor analysis. The MALDI-TOF mass spectrometer used was a Finnigan MATVision 2000 (Finnigan MAT, Bremen, Germany). Spectra were recorded inthe positive ion reflectron mode with a 5 keV ion source and 20 keVpostacceleration. The instrument was equipped with a nitrogen laser (337nm wavelength). The vacuum of the system was 3-4.10⁻⁸ hPa in theanalyzer region and 1-4.10⁻⁷ hPa in the source region. Spectra ofmodified and unmodified DNA samples were obtained with the same relativelaser power; external calibration was performed with a mixture ofsynthetic oligodeoxynucleotides (7- to 50-mer).

Results and Discussion

Enzymatic Synthesis of 7-deazapurine-nucleotide-containing Nucleic Acidsby PCR

In order to demonstrate the feasibility of MALDI-TOF MS for the rapid,gel-free analysis of short PCR products and to investigate the effect of7-deazapurine modification of nucleic acids under MALDI-TOF conditions,two different primer-template systems were used to synthesize DNAfragments. Sequences are displayed in FIGS. 36 and 37. While the twosingle strands of the 103-mer PCR product had nearly equal masses (▴M=8u), the two single strands of the 99-mer differed by 526 u.

Considering that 7-deaza purine nucleotide building blocks for chemicalDNA synthesis are approximately 160 times more expensive than regularones (Product Information, Glen Research Corporation, Sterling, Va.) andtheir application in standard β-cyano-phosphoamidite chemistry is nottrivial (Product Information, Glen Research Corporation, Sterling, Va.;Schneider, K. and B. T. Chait, Nucleic Acids Res. 23: 1570 (1995)) thecost of 7-deaza-purine-modified primers would be very high. Therefore,to increase the applicability and scope of the method, all PCRs wereperformed using unmodified oligonucleotide primers which are routinelyavailable. Substituting dATP and dGTP by c⁷-dATP and c⁷-dGTP inpolymerase chain reaction led to products containing approximately 80%7-deaza-purine-modified nucleosides for the 99-mer and 103-mer; andabout 90% for the 200-mer, respectively. Table I shows the basecomposition of all PCR products.

TABLE I Base composition of the 99-mer, 103-mer and 200-mer PCRamplification products (unmodified and 7-deaza purine modified) rel.DNA-fragments¹ C T A G c⁷-deaza-A c⁷-deaza-6 mod.² 200-mers 54 34 56 56— — — modified 200-mer 54 34 6 5 50 51 90% s 200-mer a 56 56 34 54 — — —modified 200-mer 56 56 3 4 31 50 92% a 103-mer s 28 23 24 28 — — —modified 103-mer 28 23 6 5 18 23 79% s 103-mer a 28 24 23 28 — — —modified 103-mer 28 24 7 4 16 24 78% a 99-mer s 34 21 24 20 — — —modified 99-mer 34 21 6 5 18 15 75% s 99-mer a 20 24 21 34 — — —modified 99-mer 20 24 3 4 18 30 87% a ¹“s” and “a” describe “sense” and“antisense” strands of the double-stranded amplified product. ²indicatesrelative modification as percentage of 7-deaza-purine-modifiednucleotides of total amount of purine nucleotides.

However, it remained to be determined whether 80-90% 7-deaza-purinemodification is sufficient for accurate mass spectrometer detection. Itwas therefore important to determine whether all purine nucleotidescould be substituted during the enzymatic amplification step. This wasnot trivial since it had been shown that c⁷-dATP cannot fully replacedATP in PCR if Taq DNA polymerase is employed (Seela, F. and A.Roelling, Nucleic Acids Res. 20: 55-61 (1992)). Fortunately we foundthat exo(−)Pfu DNA polymerase indeed could accept c⁷-dATP and c⁷-dGTP inthe absence of unmodified purine triphosphates. However, theincorporation was less efficient leading to a lower yield of PCR product(FIG. 38). Ethidium-bromide stains by intercalation with the stackedbases of the DNA-doublestrand. Therefore lower band intensities in theethidium-bromide stained gel might be artifacts since the modifiedDNA-strands do not necessarily need to give the same band intensities asthe unmodified ones.

To verify these results, the PCRs with [³²P]-labeled primers wererepeated. The autoradiogram (FIG. 39) clearly shows lower yields for themodified PCR-products. The bands were excised from the gel and counted.For all PCR products the yield of the modified nucleic acids was about50%, referring to the corresponding unmodified amplification product.Further experiments showed that exo(−)DeepVent and Vent DNA polymerasewere able to incorporate c⁷-dATP and c⁷-dGTP during PCR as well. Theoverall performance, however, turned out to be best for the exo(−)PfuDNA polymerase giving the least side products during amplification.Using all three polymerases, if was found that such PCRs employingc⁷-dATP and c⁷-dGTP instead of their isosteres showed fewerside-reactions giving a cleaner PCR-product. Decreased occurrence ofamplification side products may be explained by a reduction of primermismatches due to a lower stability of the complex formed from theprimer and the 7-deaza-purine-containing template which is synthesizedduring PCR. Decreased melting point for DNA duplexes containing7-deaza-purine have been described (Mizusawa, S. et al., Nucleic AcidsRes. 14: 1319-1324 (1986)). In addition to the three polymerasesspecified above (exo(−) Deep Vent DNA polymerase, Vent DNA polymeraseand exo(−) (Pfu) DNA polymerase), it is anticipated that otherpolymerases, such as the Large Klenow fragment of E.coli DNA polymerase,Sequenase, Taq DNA polymerase and U AmpliTaq DNA polymerase can be used.In addition, where RNA is the template, RNA polymerases, such as the SP6or the T7 RNA polymerase, must be used.

MALDI-TOF Mass Spectrometry of Modified and Unmodified PCR Products

The 99-mer, 103-mer and 200-mer PCR products were analyzed by MALDI-TOFMS. Based on past experience, it was known that the degree ofdepurination depends on the laser energy used for desorption andionization of the analyte. Since the influence of 7-deazapurinemodification on fragmentation due to depurination was to beinvestigated, all spectra were measured at the same relative laserenergy.

FIGS. 40A and 40B show the mass spectra of the modified and unmodified103-mer nucleic acids. In case of the modified 103-mer, fragmentationcauses a broad (M+H)⁺ signal. The maximum of the peak is shifted tolower masses so that the assigned mass represents a mean value of the(M+H)⁺ signal and signals of fragmented ions, rather than the (M+H)⁺signal itself. Although the modified 103-mer still contains about 20% Aand G from the oligonucleotide primers, it shows less fragmentationwhich is featured by much more narrow and symmetric signals. Peaktailing, especially on the lower mass side due to depurination, issubstantially reduced. Hence, the difference between measured andcalculated mass is strongly reduced although it is still below theexpected mass. For the unmodified sample, a (M+H)⁺ signal of 31670 wasobserved, which is a 97 u or 0.3% difference to the calculated mass,while in the case of the modified sample, this mass differencediminished to 10 u or 0.03% (31713 u found, 31723 u calculated). Theseobservations are verified by a significant increase in mass resolutionof the (M+H)⁺ signal of the two signal strands (m/Δm=67 as opposed to 18for the unmodified sample with Δm=full width at half maximum, fwhm).Because of the low mass difference between the two single strands (8u),their individual signals were not resolved.

With the results of the 99 base pair DNA fragments, the effects ofincreased mass resolution for 7-deazapurine-containing DNA becomes evenmore evident. The two single strands in the unmodified sample were notresolved even though the mass difference between the two strands of thePCR product was very high with 526 u due to unequal distribution ofpurines and pyrimidines (FIG. 41A). In contrast to this, the modifiedDNA showed distinct peaks for the two single strands (FIG. 41B) whichmakes the superiority of this approach for the determination ofmolecular weights to gel electrophoretic methods even more profound.Although base line resolution was not obtained, the individual masseswere able to be assigned with an accuracy of 0.1% (Δm=27 u for thelighter (calc. mass=30224 u) and Δm=14 u for the heavier strand (calc.mass=30750 u)). Again, it was found that the full width at half maximumwas substantially decreased for the 7-deazapurine containing sample.

In case of the 99-mer and the 103-mer, the 7-deazapurine containingnucleic acids seem to give higher sensitivity despite the fact that theystill contain about 20% unmodified purine nucleotides. To get comparablesignal-to-noise ratio at similar intensities for the (M+H)⁺ signals, theunmodified 99-mer required 20 laser shots in contrast to 12 for themodified one and the 103-mer required 12 shots for the unmodified sampleas opposed to three for the 7-deazapurine nucleoside-containing PCRproduct.

Comparing the spectra of the modified and unmodified 200-mer amplicons,improved mass resolution was again found for the7-deaza-purine-containing sample as well as increased signal intensities(FIGS. 42A and 42B). While the signal of the single strands predominatesin the spectrum of the modified sample the DNA-duplex and dimers of thesingle strands gave the strongest signal for the unmodified sample.

A complete 7-deaza purine modification of nucleic acids may be achievedeither using modified primers in PCR or cleaving the unmodified primersfrom the partially modified PCR product. Since disadvantages areassociated with modified primers, as described above, a 100-mer wassynthesized using primers with a ribo-modification. The primers werecleaved hydrolytically with NaOH according to a method developed earlierin our laboratory (Köster et al., Z. Physiol. Chem. 359: 1579-1589(1978)). Both hydrolyzed PCR product as well as the two released primerscould be detected, together with a small signal from residual uncleaved100-mer. This procedure is especially useful for the MALDI-TOF analysisof very short PCR-products since the share of unmodified purinesoriginating from the primer increases with decreasing length of theamplified sequence.

The remarkable properties of 7-deazapurine modified nucleic acids can beexplained by either more effective desorption and/or ionization,increased ion stability and/or a lower denaturation energy of the doublestranded purine- modified nucleic acid. The exchange of the N-7 for amethine group results in the loss of one acceptor for a hydrogen bondwhich influences the ability of the nucleic acid to form secondarystructures due to non-Watson-Crick base pairing (Seela, F. and A. Kehne,Biochemistry 26: 2232-2238 (1987)), which should be a reason for betterdesorption during the MALDI process. In addition to this, the aromaticsystem of 7-deazapurine has a lower electron density that weakensWatson-Crick base pairing resulting in a decreased melting point(Mizusawa, S. et al., Nucleic Acids Res. 14: 1319-1324 (1986)) of thedouble-strand. This effect may decrease the energy needed fordenaturation of the duplex in the MALDI process. These aspects as wellas the loss of a site which probably will carry a positive charge on theN-7 nitrogen renders the 7-deazapurine-modified nucleic acid less polarand may promote the effectiveness of desorption.

Because of the absence of N-7 as proton acceptor and the decreasedpolarization of the C—N bond in 7-deazapurine nucleosides, depurinationfollowing the mechanisms established for hydrolysis in solution isprevented. Although a direct correlation of reactions in solution and inthe gas phase is problematic, less fragmentation due to depurination ofthe modified nucleic acids can be expected in the MALDI process.Depurination may either be accompanied by loss of charge, whichdecreases the total yield of charged species, or it may produce chargedfragmentation products, which decreases the intensity of thenon-fragmented molecular ion signal.

The observation of both increased sensitivity and decreased peak tailingof the (M+H)⁺ signals on the lower mass side due to decreasedfragmentation of the 7-deazapurine-containing samples indicate that theN-7 atom indeed is essential for the mechanism of depurination in theMALDI-TOF process. In conclusion, 7-deazapurine-containing nucleic acidsshow distinctly increased ion-stability and sensitivity under MALDI-TOFconditions and therefore provide for higher mass accuracy and massresolution.

EXAMPLE 9 Solid State Sequencing and Mass Spectrometer DetectionMaterials and Methods

Oligonucleotides were purchased from Operon Technologies (Alameda,Calif.) in an unpurified form. Sequencing reactions were performed on asolid surface using reagents from the sequencing kit for SequenaseVersion 2.0 (Amersham, Arlington Heights, Ill.).

Sequencing a 39-mer Target

Sequencing Complex

5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(b))_(a)-3′(DNA11683)  (SEQ.ID.No. 23)

3′-TCAACACTGCATGT-5′ (PNA16/DNA)  (SEQ.ID.No. 24)

In order to perform solid-state DNA sequencing, template strand DNA11683was 3′-biotinylated by terminal deoxynucleotidyl transferase. A 30 μlreaction, containing 60 pmol of DNA11683, 1.3 nmol of biotin 14-dATP(GIBCO BRL, Grand Island, N.Y.), 30 units of terminal transferase(Amersham, Arlington Heights, Ill.), and 1× reaction buffer (suppliedwith enzyme), was incubated at 37° C. for 1 hour. The reaction wasstopped by heat inactivation of the terminal transferase at 70° C. for10 min. The resulting product was desalted by passing through a TE-10spin column (Clontech). More than one molecule of biotin-14-dATP couldbe added to the 3′-end of DNA11683. The biotinylated DNA11683 wasincubated with 0.3 mg of Dynal streptavidin beads in 30 μl 1× bindingand washing buffer at ambient temperature for 30 min. The beads werewashed twice with TE, resuspended in 30 μl TE, and a 10 μl aliquot(containing 0.1 mg of beads) was used for sequencing reactions.

The 0.1 mg beads from previous step were resuspended in a 10 μl volumecontaining 2 μl of 5× Sequenase buffer (200 mM Tris-HCl, pH 7.5, 100 mMMgCl₂, and 250 mM NaCl) from the Sequenase kit and 5 pmol ofcorresponding primer PNA16/DNA. The annealing mixture was heated to 70°C. and allowed to cool slowly to room temperature over a 20-30 min. timeperiod. Then 1 μl 0.1 M dithiothreitol solution, 1 μl Mn buffer (0.15 Msodium isocitrate and 0.1 M MnCl₂), and 2 μl of diluted Sequenase (3.25units) were added. The reaction mixture was divided into four aliquotsof 3 μl each and mixed with termination mixes (each consists of 3 μl ofthe appropriate termination mix: 32 μM c7dATP, 32 μM dCTP, 32 μM c7dGTP,32 μM dTTP and 3.2 μM of one of the four ddTNPS, in 50 mM NaCl). Thereaction mixtures were incubated at 37° C. for 2 min. After thecompletion of extension, the beads were precipitated and the supernatantwas removed. The beads were washed twice and resuspended in TE and keptat 4° C.

Sequencing a 78-mer Target

Sequencing Complex

5′-AAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(A^(b))_(n)-3′  (SEQ.ID.No.25)

(TNR.PLASM2)

3′-CTACTAGGCTGCGTAGTC-5′(CM1)  (SEQ.ID.No. 26)

The target TNR.PLASM2 was biotinylated and sequenced using proceduressimilar to those described in previous section (sequencing a 39-mertarget).

Sequencing a 15-mer Target with Partially-duplex Probe

Sequencing Complex

5′-F-GATGATCCGACGCATCACAGCTC-3′  (SEQ.ID.No. 27)

3′-b-CTACTAGGCTGCGTAGTGTCGAGAACCTTGGCT-5′  (SEQ.ID.No. 28)

CM1B3B was immobilized on DynaBeads M280 with streptavidin (Dynal,Norway) by incubating 60 pmol of CM1B3B with 0.3 magnetic beads in 30 μlM NaCl and TE (1× binding and washing buffer) at room temperature for 30min. The beads were washed twice with TE, resuspended in 30 μl TE, and a10 or 20 μl aliquot (containing 0.1 or 0.2 mg of beads respectively) wasused for sequencing reactions.

The duplex was formed by annealing a corresponding aliquot of beads fromprevious step with 10 pmol of DF11a5F (or 20 pmol of DF11a5F for 0.2 mgof beads) in a 9 μl volume containing 2 μl of 5x mM NaCl) from theSequenase kit. The annealing mixture was heated to 65° C. and allowed tocool slowly to 37° C. over a 20-30 min time period. The duplex primerwas then mixed with 10 pmol of TS10 (20 pmol of TS10 for 0.2 mg ofbeads) in 1 μl volume, and the resulting mixture was further incubatedat 37° C. for 5 min., and room temperature for 5-10 min. Then 1 μl 0.1 Mdithiothreitol solution, 1 μl Mn buffer (0.15 M sodium isocitrate and0.1 M MnCl₂), and 2 μl of diluted Sequenase (3.25 units) were added. Thereaction mixture was divided into four aliquots of 3 μl each and mixedwith termination mixes (each consists of 4 μl of the appropriatetermination mix: 16 μM dATP, 16 μM dCTP, 16 μM dGTP, 16 μM dTTP and 1.6μM of one of the four ddNTPs, in 50 mM NaCl). The reaction mixtures wereincubated at room temperature for 5 min., and 37° C. for 5 min. Afterthe completion of extension, the beads were precipitated and thesupernatant was removed. The beads were resuspended in 20 μl TE and keptat 4° C. An aliquot of 2 μl (out of 20 μl ) from each tube was taken andmixed with 8 μl of formamide, the resulting samples were denatured at90-95° C. for 5 min. and 2 μl (out of 1 0 μl total) was applied to anALF DNA sequencer (Pharmacia, Piscataway, N.J.) using a 10%polyacrylamide gel containing 7 M urea and 0.6×TBE. The remainingaliquot was used for MALDI-TOF MS analysis.

MALDI Sample Preparation and Instrumentation

Before MALDI analysis, the sequencing-ladder-loaded magnetic beads werewashed twice using 50 mM ammonium citrate and resuspended in 0.5 μl purewater. The suspension was then loaded onto the sample target of the massspectrometer and 0.5 μl of saturated matrix solution (3-hydropicolinicacid (HPA): ammonium citrate=10:1 mole ratio in 50% acetonitrile) wasadded. The mixture was allowed to dry prior to mass spectrometeranalysis.

The reflectron TOFMS mass spectrometer (Vision 2000, Finnigan MAT,Bremen, Germany) was used for analysis. 5 kV was applied in the ionsource and 20 kV was applied for postaccelaration. All spectra weretaken in the positive ion mode and a nitrogen laser was used. Normally,each spectrum was averaged for more than 100 shots and a standard25-point smoothing was applied.

Results and Discussion

Conventional Solid-state Sequencing

In conventional sequencing methods, a primer is directly annealed to thetemplate and then extended and terminated in a Sanger dideoxysequencing. Normally, a biotinylated primer is used and the sequencingladders are captured by streptavidin-coated magnetic beads. Afterwashing, the products are eluted from the beads using EDTA andformamide. However, our previous findings indicated that only theannealed strand of a duplex is desorbed and the immobilized strandremains on the beads. Therefore, it is advantageous to immobilize thetemplate and anneal the primer. After the sequencing reaction andwashing, the beads with the immobilized template and annealed sequencingladder can be loaded directly onto the mass spectrometer target andmixed with matrix. In MALDI, only the annealed sequencing ladder will bedesorbed and ionized, and the immobilized template will remain on thetarget.

A 39-mer template (SEQ.ID.No. 23) was first biotinylated at the 3′ endby adding biotin-14-dATP with terminal transferase. More than onebiotin-14-dATP molecule could be added by the enzyme. However, since thetemplate was immobilized and remained on the beads during MALDI, thenumber of biotin-14-dATP added would not affect the mass spectra. A14-mer primer (SEQ.ID.No. 29) was used for the solid-state sequencing.MALDI-TOF mass spectra of the four sequencing ladders are shown in FIGS.44A-44D and the expected theoretical values are shown in Table II.

TABLE II  1 5′-TCTGGCCTGGTGCAGGGCCTATTGTAGTTGTGACGTACA-(A^(B))_(n)-3′  23′-TCAACACTGCATGT-5-    3 3′-ATCAACACTGCATGT-5′    43′-CATCAACACTGCATGT-5′    5 3′-ACATCAACACTGCATGT-5′    63′-AACATCAACACTGCATGT-5′    7 3′-TAACATCAACACTGCATGT-5′    83′-ATAACATCAACACTGCATGT-5′    9 3′-GATAACATCAACACTGCATGT-5′   103′-GGATAACATCAACACTGCATGT-5′   11 3′-CGGATAACATCAACACTGCATGT-5′   123′-CCGGATAACATCAACACTGCATGT-5′   13 3′-CCCGGATAACATCAACACTGCATGT-5′   143′-TCCCGGATAACATCAACACTGCATGT-5′   153′-GTCCCGGATAACATCAACACTGCATGT-5′   163′-CGTCCCGGATAACATCAACACTGCATGT-5′   173′-ACGTCCCGGATAACATCAACACTGCATGT-5′   183′-CACGTCCCGGATAACATCAACACTGCATGT-5′   193′-CCACGTCCCGGATAACATCAACACTGCATGT-5′   203′-ACCACGTGCCGGATAACATCAACACTGCATGT-5′   213′-GACCACGTGCCGGATAACATCAACACTGCATGT-5′   223′-GGACCACGTCCCGGATAACATCAACACTGCATGT-5′   233′-CGGACCACGTCCCGGATAACATCAACACTGCATGT-5′   243′-CGGGACCACGTCCCGGATAACATCAACACTGCATGT-5′   253′-ACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′   263′-GACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′   273′-AGACCGGACCACGTCCCGGATAACATCAACACTGCATGT-5′   A-reaction C-reactionG-reaction T-reaction 1. 2. 4223.8 4223.8 4223.8 4223.8 3. 4521.1 4.4810.2 5. 5123.4 6. 5436.6 7. 5740.8 8. 6054.0 9. 6383.2 10. 6712.4 11.7001.6 12. 7290.8 13. 7580.0 14. 7884.2 15. 8213.4 16. 8502.6 17. 8815.818. 9105.0 19. 9394.2 20. 9707.4 21. 10036.6 22. 10365.8 23. 10655.0 24.10944.2 25. 11257.4 26. 11586.6 27. 11899.8

The sequencing reaction produced a relatively homogenous ladder, and thefull-length sequence was determined easily. One peak around 5150appeared in all reactions was not identified. A possible explanation isthat a small portion of the template formed some kind of secondarystructure, such as a loop, which hindered Sequenase extension.Mis-incorporation is of minor importance, since the intensity of thesepeaks were much lower than that of the sequencing ladders. Although7-deaza purines were used in the sequencing reaction, which couldstabilize the N-glycosidic bond and prevent depurination, minor baselosses were still observed since the primer was not substituted by7-deazapurines. The full length ladder, with a ddA at the 3′ end,appeared in the A reaction with an apparent mass of 11899.8. However, amore intense peak of 122 appeared in all four reactions and is likelydue to an addition of an extra nucleotide by the Sequenase enzyme.

The same technique could be used to sequence longer DNA fragments. A78-mer template containing a CTG repeat (SEQ.ID.No. 25) was3′-biotinylated by adding biotin-14-dATP with terminal transferase. An18-mer primer (SEQ.ID.No. 26) was annealed right outside the CTG repeatso that the repeat could be sequenced immediately after primerextension. The four reactions were washed and analyzed by MALDI-TOF MSas usual. An example of the G-reaction is shown in FIG. 45 and theexpected sequencing ladder is shown in Table III with theoretical massvalues for each ladder component. All sequencing peaks were wellresolved except the last component (theoretical value 20577.4) wasindistinguishable from the background. Two neighboring sequencing peaks(a 62-mer and a 63-mer) were also separated indicating that suchsequencing analysis could be applicable to longer templates. Again, anaddition of an extra nucleotide by the Sequenase enzyme was observed inthis spectrum. This addition is not template specific and appeared inall four reactions which makes it easy to be identified. Compared to theprimer peak, the sequencing peaks were at much lower intensity in thelong template case. Further optimization of the sequencing reaction maybe required.

TABLE IIIAAGATCTGACCAGGGATTCGGTTAGCGTGACTGCTGCTGCTGCTGCTGGATGATCCGACGCATCAGATCTGG-(A^(B))_(n)-3′1 3′-CTACTAGGCTGCGTAGTC-5′             23′-CCTACTAGGCTGCGTAGTC-5′             33′-ACCTACTAGGCTGCGTAGTC-5′             43′-GACCTACTAGGCTGCGTAGTC-5′             53′-CGACCTACTAGGCTGCGTAGTC-5′             63′-ACGACCTACTAGGCTGCGTAGTC-5′             73′-GACGACCTACTAGGCTGCGTAGTC-5′             83′-CGACGACCTAGTAGGCTGCGTAGTC-5′             93′-ACGACGACCTACTAGGCTGCGTAGTC-5′             103′-GACGACGACCTAGTAGGCTGCGTAGTC-5′             113′-CGACGACGACCTACTAGGCTGCGTAGTC-5′             123′-ACGACGACGACCTACTAGGCTGCGTAGTC-5′             133′-GACGACGACGACCTACTAGGCTGCGTAGTC-5′             143′-CGACGACGACGACCTACTAGGCTGCGTAGTC-5′             153′-ACGACGACGACGACCTACTAGGCTGCGTAGTC5-′             163′-GACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             173′-CGACGACGACGACGAGCTACTAGGCTGCGTAGTC-5′             183′-ACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             193′-GACGAGGACGACGACGACCTACTAGGCTGCGTAGTC-5′             203′-CGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             213′-ACGACGACGACGACGACGAGCTACTAGGCTGCGTAGTC-5′             223′-GACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             233′-CGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             243′-ACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             253′-GACGACGACGACGACGACGACGACCTACTAGGGTGCGTAGTC-5′             263′-TGACGACGACGACGACGACGACGACCTACTAGGGTGCGTAGTC-5′             273′-CTGACGACGACGACGACGACGACGAGCTAGTAGGCTGCGTAGTC-5′             283′-ACTGACGACGACGACGACGACGACGAGCTACTAGGCTGCGTAGTC-5′             293′-CACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             303′-GCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             313′-CGCAGTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             323′-TCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′             333′-ATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′             343′-AATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′             353′-CAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            363′-CCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            373′-GCCAATCGCAGTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′            383′-AGCCAATCGCACTGACGACGACGACGACGACGACGACCTAGTAGGCTGGGTAGTC-5′            393′-AAGCCAATCGCACTGACGACGACGACGACGACGACGACGTACTAGGCTGCGTAGTC-5′            403′-TAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            413′-CTAAGCGAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′            423′-CCTAAGCCAATCGCAGTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTG-5′            433′-CCGTAAGCCAATCGGACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            443′-TCGCTAAGCGAATCGCACTGAGGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            453′-GTCCCTAAGCCAATCGCAGTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            463′-GGTCCGTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            473′-TGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′            483′-CTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGAGGACGACCTACTAGGCTGCGTAGTC-5′            493′-AGTGGTCGGTAAGCGAATCGCAGTGAGGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′            503′-GACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            513′-AGACTGGTCGCTAAGCCAATCGCACTGACGACGACGACGACGAGGACGACCTAGTAGGCTGCGTAGTC-5′            523′-TAGACTGGTCCCTAAGCCAATCGCAGTGACGACGACGACGACGACGACGACCTAGTAGGCTGGGTAGTC-5′            533′-CTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGGGTAGTC-5′            543′-TCTAGACTGGTCCCTAAGCCAATCGCAGTGACGAGGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            553′-TTCTAGACTGGTCCCTAAGCCAATCGCACTGACGACGACGACGACGACGACGACCTACTAGGCTGCGTAGTC-5′            ddATP ddCTP ddGTP ddTTP 1. 5491.6 5491.6 5491.6 5491.6 2. 5764.8 3.6078.0 4. 6407.2 5. 6696.4 6. 7009.6 7. 7338.8 8. 7628.0 9. 7941.2 10.8270.4 11. 8559.6 12. 8872.8 13. 9202.0 14. 9491.2 15. 9804.4 16.10133.6 17. 10422.88 18. 10736.0 19. 11065.2 20. 11354.4 21. 11667.6 22.11996.8 23. 12286.0 24. 12599.2 25. 12928.4 26. 13232.6 27. 13521.8 28.13835.0 29. 14124.2 30. 14453.4 31. 14742.6 32. 15046.8 33. 15360.0 34.15673.2 35. 15962.4 36. 16251.6 37. 16580.8 38. 16894.0 39. 17207.2 40.17511.4 41. 17800.6 42. 18189.8 43. 18379.0 44. 18683.2 45. 19012.4 46.19341.6 47. 19645.8 48. 19935.0 49. 20248.2 50. 20577.4 51. 20890.6 52.21194.4 53. 21484.0 54. 21788.2 55. 22092.4

Sequencing Using Duplex DNA Probes for Capturing and Priming

Duplex DNA probes with single-stranded overhang have been demonstratedto be able to capture specific DNA templates and also served as primersfor sold-state sequencing. The scheme is shown in FIG. 46. Stackinginteractions between a duplex probe and a single-stranded template allowonly 5-base overhang to be to be sufficient for capturing. Based on thisformat, a 5′ fluorescent-labeled 23-mer (5′-GAT GAT CCG ACG CAT CAC AGCTC-3′) (SEQ.ID.No. 29)) was annealed to a 3′-biotinylated 18-mer (5′-GTGATG CCT CGG ATC ATC-3′) (SEQ.ID.No. 30)), leaving a 5-base overhang. A15-mer template (5′-TCG GTT CCA AGA GCT-3′) (SEQ.ID.No. 31)) wascaptured by the duplex and sequencing reactions were performed byextension of the 5-base overhang. MALDI-TOF mass spectra of thereactions are shown in FIGS. 47A-47D. All sequencing peaks wereresolved, although at relatively low intensities. The last peak in eachreaction is due to unspecific addition of one nucleotide to the fulllength extension product by the Sequenase enzyme. For comparison, thesame products were run on a conventional DNA sequencer and a stackingfluorogram of the results is shown in FIGS. 48A-48D. As can be seen fromthe Figures, the mass spectra had the same pattern as the fluorogramwith sequencing peaks at much lower intensity compared to the 23-merprimer.

Improvements of MALDI-TOF Mass Spectrometry as a Dection Technique

Sample distribution can be made more homogenous and signal intensitycould potentially be increased by implementing the picoliter vialtechnique. In practice, the samples can be loaded on small pits withsquare openings of 100 μm size. The beads used in the solid statesequencing are less than 10 μm in diameter, so they should fit well inthe microliter vials. Microcrystals of matrix and DNA containing “sweetspots” will be confined in the vial. Since the laser spot size is about100 μm in diameter, it will cover the entire opening of the vial.Therefore, searching for sweet spots will be unnecessary and highrepetition-rate laser (e.g. <=>10Hz) can be used for acquiring spectra.An earlier report has shown that this device is capable of increasingthe detection sensitivity of peptides and proteins by several orders ofmagnitude compared to the conventional MALDI sample preparationtechnique.

Resolution of MALDI on DNA needs to be further improved in order toextend the sequencing range beyond 100 bases. Currently, using3-HPA/ammonium citrate as matrix and a reflectron TOF mass spectrometerwith 5 kV ion source and 20 kV postacceleration, the resolution of therun-through peak in FIG. 33A (73-mer) is greater than 200 (FWHM) whichis enough for sequence determination in this case. This resolution isalso the highest reported for MALDI desorbed DNA ions above the 70-merrange. Use of the delayed extraction technique may further enhanceresolution.

All of the above-cited references, applications and publications areherein incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

33 20 base pairs nucleic acid single linear cDNA 1 GCAAGTGAAT CCTGAGCGTG20 19 base pairs nucleic acid single linear cDNA 2 GTGTGAAGGG TTCATATGC19 28 base pairs nucleic acid single linear cDNA 3 ATCTATATTC ATCATAGGAAACACCACA 28 30 base pairs nucleic acid single linear cDNA 4 GTATCTATATTCATCATAGG AAACACCATT 30 30 base pairs nucleic acid single linear cDNA 5GCTTTGGGGC ATGGACATTG ACCCGTATAA 30 30 base pairs nucleic acid singlelinear cDNA 6 CTGACTACTA ATTCCCTGGA TGCTGGGTCT 30 20 base pairs nucleicacid single linear cDNA 7 TTGCCTGAGT GCAGTATGGT 20 20 base pairs nucleicacid single linear cDNA 8 AGCTCTATAT CGGGAAGCCT 20 24 base pairs nucleicacid single linear cDNA 9 TTGTGCCACG CGGTTGGGAA TGTA 24 26 base pairsnucleic acid single linear cDNA 10 AGCAACGACT GTTTGCCCGC CAGTTG 26 25base pairs nucleic acid single linear cDNA 11 TACATTCCCA ACCGCGTGGCACAAC 25 25 base pairs nucleic acid single linear cDNA 12 AACTGGCGGGCAAACAGTCG TTGCT 25 57 base pairs nucleic acid single linear cDNA 13ACCATTAAAG AAAATATCAT CTTTGGTGTT TCCTATGATG AATATAGAAG CGTCATC 57 24base pairs nucleic acid single linear cDNA 14 ACCACAAAGG ATACTACTTA TATC24 29 base pairs nucleic acid single linear cDNA 15 TAGAAACCACAAAGGATACT ACTTATATC 29 26 base pairs nucleic acid single linear cDNA 16TAACCACAAA GGATACTACT TATATC 26 29 base pairs nucleic acid single linearcDNA 17 TAGAAACCAC AAAGGATACT ACTTATATC 29 38 base pairs nucleic acidsingle linear cDNA 18 CTTTTATAGT AGAAACCACA AAGGATACTA CTTATATC 38 35base pairs nucleic acid single linear cDNA 19 CTTTTATAGT AACCACAAAGGATACTACTT ATATC 35 35 base pairs nucleic acid single linear cDNA 20CTTTTATAGA AACCACAAAG GATACTACTT ATATC 35 31 base pairs nucleic acidsingle linear cDNA 21 CGTAGAAACC ACAAAGGATA CTACTTATAT C 31 58 basepairs nucleic acid single linear cDNA 22 GAATTACATT CCCAACCGCGTGGCACAACA ACTGGCGGGC AAACAGTCGT TGCTGATT 58 58 base pairs nucleic acidsingle linear cDNA 23 AATCAGCAAC GACTGTTTGC CCGCCAGTTG TTGTGCCACGCGGTTGGGAA TGTAATTC 58 252 base pairs nucleic acid single linear cDNA 24GGCACGGCTG TCCAAGGAGC TGCAGGCGGC GCAGGCCCGG CTGGGCGCGG ACATGGAGGA 60CGTGTGCGCC GCCTGGTGCA GTACCGCGGC GAGGTGCAGG CCATGCTCGG CCAGAGCAC 120GAGGAGCTGC GGGTGCGCCT CGCCTCCCAC CTGCGCAAGC TGCGTAAGCG GCTCCTCCG 180GATGCCGATG ACCTGCAGAA GTCCCTGGCA GTGTACCAGG CCGGGGCCCG CGAGGGCGC 240GAGCGCGGCC TC 252 110 base pairs nucleic acid single linear cDNA 25GCAACATTTT GCTGCCGGTC ACGGTTCGAA CGTACGGACG TCCAGCTGAG ATCTCCTAGG 60GGCCCATGGC TCGAGCTTAA GCATTAGTAC CAGTATCGAC AAAGGACACA 110 110 basepairs nucleic acid single linear cDNA 26 TGTGTCCTTT GTCGATACTGGTACTAATGC TTAAGCTCGA GCCATGGGCC CCTAGGAGAT 60 CTCAGCTGGA CGTCCGTACGTTCGAACCGT GACCGGCAGC AAAATGTTGC 110 217 base pairs nucleic acid singlelinear cDNA 27 AACGTGCTGC CTTCCACCGC GATGTTGATG ATTATGTGTC TGAATTTGATGGGGGCAGGC 60 GGCCCCCGTC TGTTTGTCGC GGGTCTGGTG TTGATGGTGG TTTCCTGCCTTGTCACCCT 120 GACCTGCAGC CCAAGCTTGG GATCCACCAC CATCACCATC ACTAATAATGCATGGGCTG 180 AGCCAATTGG CACTGGCCGT CGTTTTACAA CGTCGTG 217 217 basepairs nucleic acid single linear cDNA 28 CACGACGTTG TAAAACGACGGCCAGTGCCA ATTGGCTGCA GCCCATGCAT TATTAGTGAT 60 GGTGATGGTG GTGGATCCCAAGCTTGGGCT GCAGGTCGAG GGTGACAAGG CAGGAAACC 120 CCATCAACAC CAGACCCGCGACAAACAGAC GGGGGCCGCC TGCCCCCATC AAATTCAGA 180 ACATAATCAT CAACATCGCGGTGGAAGGCA GCACGTT 217 17 base pairs nucleic acid single linear cDNA 29GTAAAACGAC GGCCAGT 17 17 base pairs nucleic acid single linear cDNA 30CAGGAAACAG CTATGAC 17 18 base pairs nucleic acid single linear cDNA 31CTTCCACCGC GATGTTGA 18 19 base pairs nucleic acid single linear cDNA 32TTGTAAAACG ACGGCCAGT 19 18 base pairs nucleic acid single linear cDNA 33GTCACCCTCG ACCTGCAG 18

What is claimed is:
 1. A solid support, comprising: a plurality ofsingle-stranded oligonucleotides immobilized thereon by a linkercleavable under conditions of matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry; and matrix forperforming MALDI mass spectrometry, wherein the support comprises a flatsurface.
 2. The solid support of claim 1, wherein the oligonucleotidescomprise an array.
 3. The solid support of claim 1 that is selected fromthe group consisting of glass fiber filters, glass surfaces and metalsurfaces.
 4. The solid support of claim 1 that is selected from thegroup consisting of steel, gold, silver, aluminum, copper and silicon.5. The solid support of claim 1 that is a silicon.
 6. The solid supportof claim 1 that is a silicon wafer.
 7. The solid support of claim 1,wherein the linker is photocleavable.
 8. The solid support of claim 1,wherein the oligonucleotides comprise oligoribonucleotides,oligodeoxyribonucleotides, nucleotide analogs, or peptide nucleic acids(PNA).
 9. The solid support of claim 8, wherein the nucleotide analogscomprise a thio-modified phosphodiester or phosphotriester backbone. 10.The support of claim 1, wherein the linker is selected from the groupconsisting of a heterobifunctional derivative of a trityl ether group, acharge transfer complex and a quaternary ammonium group.
 11. The solidsupport of claim 1, wherein an oligonucleotide spacer separates eacholigonucleotide strand from the support.
 12. The solid support of claim11, wherein the oligonucleotide spacer is at least about fivenucleotides in length.
 13. A solid support, comprising: a plurality ofsingle-stranded oligonucleotides immobilized thereon by a linkerselected from the group consisting of a leuvinyl group, a pyrophosphatebond, and a ribonucleotide bond; and matrix for performing MALDI massspectrometry, wherein the support comprises a flat surface.